NEUROBIOLOGY

PROTOCOL HANDBOOK

Make the connection

Contents
Introduction 5
1 Neural development and neural stem cells 5
2 Neural cell types in neurological diseases 9

Neural cell culture and differentiation 13

3 Recovery, culture, and characterization of cortical and hippocampal neurons 13
4 Thawing and culture of cryopreserved neurons 17
5 Culturing human fetal neural stem cells 20
6 Culturing rat fetal neural stem cells 24
7 Differentiating glial precursor cells into astrocytes and oligodendrocytes 28
8 Xeno-free culture of neural stem cells 32
9 Creation of neural stem cells from human pluripotent stem cells 35
10 Differentiating human pluripotent stem cell–derived neural stem cells into neurons 40
11 PSC-derived neuron cell culture: Neuronal differentiation, maturation, and maintenance 44
12 Differentiating PSCs to midbrain dopaminergic neurons 50
13 Culture of cortical astrocytes 58
14 Cryopreserving neural stem cells 65

Cell analysis 73
15 Cell viability assays for neurons and neural cells 73
16 Markers for characterizing neural subtypes 76
17 Flow cytometry analysis 78
18 Immunocytochemistry 80
19 Quantitative image analysis 86
20 Intracellular calcium assay using fluo-4 90
21 Measuring membrane potential using the FluoVolt kit 92

Molecular characterization 95
22 PCR primers for molecular characterization of neural subtypes 95
23 RNA isolation and cDNA preparation from neural cells 96
24 Characterizing neural cells by qPCR 99

Transfection 103
25 Lipid-mediated transfection of neuronal cells 103
26 Transfecting neural cells using the Neon Transfection System 114

Appendix 123
A1 Thermo Fisher Scientific products 123
A2 Resources for more information 133
A3 Technical support 135
4
Introduction
1 Neural development and neural stem cells

Overview

It has long been thought that the adult mammalian nervous pluripotent stem cells (iPSCs), have the capacity for
system is incapable of regeneration after injury. However, multilineage differentiation, enabling construction of a
recent advances in our understanding of stem cell biology complete, viable organism (i.e., they are pluripotent).
and neuroscience have opened up new avenues of In contrast, adult stem cells can generate only one
research for developing potential treatments for incurable specific lineage of differentiated cells to reconstitute
neurodegenerative diseases and neuronal injuries. Because tissues or organs.
stem cells have the capacity to self-renew and generate
differentiated cells, stem cell replacement therapy for Neural stem cells
central and peripheral nervous system disorders and Neural stem cells (NSCs) are stem cells in the nervous
injuries strives to repopulate the affected neural tissue system that can self-renew and give rise to differentiated
with neurons and other neural cells. One of the main progenitor cells that generate lineages of neurons as well
strategies towards this end aims to recapitulate the normal as glia, such as astrocytes and oligodendrocytes. This
development of the nervous system by activating the characteristic is known as multipotency. NSCs and neural
endogenous regenerative capacity of neural stem cells or progenitor cells are present throughout development and
by transplanting neural or embryonic cells. persist in the adult nervous system. Multiple classes of
NSCs have been identified that differ from each other in
This chapter defines the key concepts in stem cell biology their differentiation abilities, their cytokine responses, and
with respect to the nervous system, presents an overview their surface antigen characteristics.
of neural development, and summarizes the involvement of
neural cell types in specific neural diseases. Rationale for studying neural stem cells
Neurological disorders, especially neurodegenerative
disorders, are at the top of the list of diseases that have
Stem cells been suggested as targets for stem cell therapy. A
thorough characterization of NSCs, a better understanding
The classical definition of a stem cell describes the of neural patterning, and the generation of all three major
capacity to self-renew and that it possesses potency. cell types that constitute the central nervous system (i.e.,
Self-renewal is defined as the ability of the stem cell to go neurons, astrocytes, and oligodendrocytes), as well as the
through multiple cycles of cell division while maintaining its microenvironments that can support them, are crucial to
undifferentiated state (i.e., to generate daughter cells that increase the likelihood of clinical success in the use of stem
are identical to the mother cell). Potency is the ability of the cells in neurological disorders.
stem cell to differentiate into specialized cell types.
Stem cells and cancer
Pluripotent vs. adult stem cells The discovery that many cancers may be propagated by a
A stem cell can divide to generate one daughter cell that small number of stem cells present in a tumor mass is an
is a stem cell, maintaining its capacity for self-renewal extremely exciting finding. This was first described in breast
and potency, and another daughter cell that can further cancers and subsequently in a variety of solid tumors.
produce differentiated cells. Some pluripotent stem Several reports have suggested that cancer stem cells
cells, including embryonic stem cells (ESC) and induced can be identified in the nervous system as well, and that

5
Introduction

these cells bear a remarkable similarity to neural stem cells neural progenitor cells; this stage in development is called
present in early development. Likewise, cells resembling neural ectoderm induction. By studying neural induction
glial progenitors have been isolated from some glial tumors, and neural development, we can determine the various
suggesting an intriguing link between developmental factors that stimulate or inhibit the differentiation of NSCs
and cancer biology. and the requirements of these NSCs and their offspring for
survival and proper function.

Neural development Stages of neural development

The nervous system is one of the earliest organ systems
Establishment of the central nervous system (CNS) is that differentiate from the blastula-stage embryo. This
initiated early in development by the induction of NSCs and differentiation can be mimicked in culture, and NSCs

Special notice about B-27 Supplement cell–derived neurons can be challenging, as these cells
For over 25 years, the classic Gibco™ B-27™ Supplement are quite sensitive and tend to undergo progressive
and Gibco™ Neurobasal™ Medium have set the standard cell death over time. The B-27 Plus Neuronal Culture
for neuronal cell culture reagents. Originally designed System improves survival by more than 50%, without
for the serum-free culture of primary neurons, B-27 the need for additional components such as serum.
Supplement and Neurobasal Medium have been widely
adapted to other applications and cell types, including, • Improve neuronal functionality—With increased
among others, PSC-derived neurons and the long-term survival comes a superior initial quality of neurons.
culture of primary neurons. Indicators of this are superior neurite outgrowth,
increased synaptic complexity, and improvement
To meet the growing needs of researchers, we have of electrophysiological activity. The B-27 Plus
optimized the nutritive properties of B-27 Supplement Neuronal Culture System has been shown to improve
and Neurobasal Medium to better support primary functionality of primary and stem cell–derived neurons.
and stem cell–derived neurons. The Gibco™ B-27™ Plus
Neuronal Culture System, composed of the Gibco™ B-27™ • Improve neuronal maturity—During maturation,
Plus Supplement and Gibco™ Neurobasal™ Plus Medium, neurons extend to form highly connected networks,
improves upon the classic culture environment through express synaptic markers, and generate spontaneous,
raw material and manufacturing upgrades and minor networked electrical activity. Robust maturation is
formulation modifications. Together, these small changes necessary for the generation of relevant disease
yield big results. model systems. The B-27 Plus Neuronal Culture
System increases synaptic complexity of neurons,
The updated protocols in this handbook include the B-27 leading to more extensive expression of pre- and
Plus Neuronal Culture System, which will: post-synaptic markers.

• Improve neuronal survival—Maintaining healthy For more information, visit

long-term cultures of primary rodent neurons and stem thermofisher.com/b27plus

6
 Introduction

can be derived from human ESC cultures over a period parallel the growth of the embryo as it undergoes further
of 1–2 weeks. In vivo, the primitive neural tube forms by differentiation and forms the spinal cord. The ventricular
approximately the fourth week of gestation via a process zone stem cells appear homogeneous despite the
termed primary neurulation. Neurogenesis commences by acquisition of rostrocaudal and dorsoventral identity,
the fifth week of development in humans. but differ in their differentiation ability and self-renewal
capacity. Specific regions of the brain may have relatively
Separation of PNS and CNS distinct stem cell populations, such as the developing
During neurulation, the neuroectoderm segregates from the retina and the cerebellum.
ectoderm, and the initially formed neural plate undergoes
a stereotypic set of morphogenetic movements to form Stem cells in the subventricular zone
a hollow tube. Neural crest stem cells generate the As development proceeds, the ventricular zone is reduced
peripheral nervous system (PNS), which segregates from in size and additional zones of mitotically active precursors
the CNS at this stage. Neural crest stem cells generate appear. Mitotically active cells that accumulate adjacent
the sympathetic and parasympathetic systems, the dorsal to the ventricular zone are called the subventricular zone
root ganglia and the cranial nerves, and the peripheral glia, (SVZ) cells. The SVZ later becomes the subependymal
including Schwann cells and enteric glia. In addition to zone as the ventricular zone is reduced to a single layer
neural derivatives, the cranial crest generates craniofacial of ependymal cells. The SVZ is prominent in the forebrain
mesenchyme that includes bone, cartilage, teeth, and and can be identified as far back as the fourth ventricle,
smooth muscle, while both cranial and caudal crests but it cannot be detected in more caudal regions of the
generate melanocytes. Placodes, which will form a subset brain; if it exists in these regions, it likely consists of a
of the peripheral nervous system and the cranial nerves, very small population of cells. An additional germinal
arise at this stage as well. These populations, which are matrix derived from the rhombic lip of the fourth ventricle,
distinct from CNS stem cells, can use similar media and called the external granule layer, generates the granule
culture conditions for propagation over limited time periods. cells of the cerebellum.

Stem cells in the ventricular zone Like the VZ, the SVZ can be divided into subdomains
Stem cells that will generate the CNS reside in the that express different rostrocaudal markers and generate
ventricular zone (VZ) throughout the rostrocaudal axis phenotypically distinct progeny. Distinct SVZ domains
and appear to be regionally specified. These stem cells include the cortical SVZ, the medial ganglion eminence,
proliferate at various rates and express different positional and the lateral ganglion eminence. The proportion of SVZ
markers. The anterior neural tube undergoes a significant stem cells declines with development, and multipotent
expansion and can be delineated into three primary stem cells are likely to be present only in regions of ongoing
vesicles: the forebrain (prosencephalon), the midbrain neurogenesis (e.g., anterior SVZ and the SVZ underlying
(mesencephalon), and the hindbrain (rhombencephalon). the hippocampus) in the adult CNS. At this stage, marker
Differential growth and further segregation leads to expression is relatively heterogeneous. Other relatively less-
additional delineation of the prosencephalon into the characterized stem cells have also been described.
telencephalon and diencephalon, and delineation of
the rhombencephalon into the metencephalon and Neural precursor cells
myelencephalon. The caudal neural tube does not Neural stem cells do not generate differentiated progeny
undergo a similar expansion, but increases in size to directly but rather generate dividing populations of more

7
Introduction

restricted precursors analogous to the blast cells, or

restricted progenitors described in hematopoietic lineages.
These precursors can divide and self-renew, but they are
located in regions distinct from the stem cell population
and can be distinguished from them by the expression of
cell surface and cytoplasmic markers and their ability to
differentiate. Several such classes of precursors have been
identified, including neuronal precursors and uni-, bi-, and
tri-potential glial precursors that generate astrocytes and
oligodendrocytes. Other precursors such as a neuron-
astrocyte precursor may also exist, and the same precursor
may have multiple names. Such precursors can be
distinguished from stem cells by their marker expression,
ability to differentiate, and time of development.

8
 Introduction

2 Neural cell types in neurological diseases

This table lists some of the better-known neurological

disorders with experimental models, cell types, growth
factors, and markers studied.

Neural Experimental Growth Progenitor Mature

Cell type Markers Transplantation Reference
disease model factor(s) cell(s) markers
Spinal cord Transplantation of Oligodendrocyte EGF, bFGF, OPC OLIG1, A2B5, GalC, MBP, Yes Keirstead
injury oligodendrocyte PDGF, RA SOX10, NG2, RIP et al., 2005
progenitor cells O4
(OPCs) into
demyelination model
Alzheimer’s Transgenic models Cholinergic BDNF Cholinergic Aβ or tau Val66Met No Giri et al.,
disease targeting APP, neuron neurons, accumulations polymorphism 2016
presenilin, tau NMDA in neurons,
proteins, tauopathy, receptors monitor
atrophy, and neural neurofibrillary
loss in frontal cortex tangles or Aβ
as well as in other peptide
regions
Multiple Demyelinated axons, Oligodendrocyte EGF, bFGF, OPC PDGFR, A2B5, O4, O1, MBP, No Kang et al.,
sclerosis co-cultured with PDGF, RA NG2 PLP 2007
rat hippocampal
neurons
Remyelination Oligodendrocyte RA, EGF, OPC PDGFR, NG2, O4, O1, MBP, Yes Izrael et al.,
models bFGF, OLIG1/2, PLP 2007
noggin, SOX10
vitamin C
Amyotrophic Transplantation of Motoneuron BDNF, GDNF, Motoneuron HOXB4, NKX6-1, Yes Lee et al.,
lateral motoneuron progeny AA, RA, progenitor NKX6-1/6-2, OLIG2, NGN2, 2007
sclerosis and into the developing SHH, noggin OLIG1/2 ISL1, ChAT,
spinal chick embryo VAChT, HB9,
muscular LHX3, HOX
atrophy In vitro studies only Motoneuron bFGF, RA, Motoneuron OLIG1/2, NKX6-1, No Li et al.,
SHH, BDNF, progenitor NKX6-1/6-2, OLIG2, NGN2, 2005
GDNF, IGF-1 NGN2 ISL1, ChAT,
VACht, HB9,
synapsin

9
Introduction

Neural Experimental Growth Progenitor Mature

Cell type Markers Transplantation Reference
disease model factor(s) cell(s) markers
Parkinson’s Not applicable Dopaminergic SHH, FGF8, DA neuron PAX2, PAX5, MAP2, TH, No Perrier et al.,
disease (DA) neuron BDNF, AA, precursor LMX, EN1 AADC, VMAT, 2004
TGFβ, TGF-3 NURR1, PTX3
In vitro drug DA neuron FGF2 or DA neuron EN1, OTX2, TH, GABA, No Yan et al.,
screening FGF8, SHH, precursor WNT1, PAX2, EN1, AADC 2005
BDNF, GDNF, GBX2
cAMP, AA
Transplantation DA neuron FGF2, FGF8, DA neuron EN1, PAX2, TH, TUJ-1 Yes Roy et al.,
into the neostriata SHH, BDNF, precursor OTX2 2006
of 6-hydroxy­ GDNF, FBS
dopamine–lesioned
Parkinsonian rats
Transplantation DA neuron SHH, FGF8, DA neuron PAX2, EN1, TH, EN1, Yes Park et al.,
into the striatum of BDNF, GDNF, precursor NURR1, AADC 2005
hemi-Parkinsonian AA, IGF-1 LMX1B
rats
Transplantation DA neuron SHH, dual Midbrain floor FoxA2, TUJ-1, TH, Yes Kriks et al.,
into the striatum of Smad plate cells LMX1A, OTX2 NURR1, 2011
hemi-Parkinsonian inhibitor, FoxA2, PTX3
rats and MPTP- FGF8, CHIR,
treated rhesus BDNF, GDNF,
monkeys dbcAMP,
TGFb3
Glial-related Astrocyte-related Astrocyte Cyclopamine, — — GFAP, S100, No Lee et al.,
diseases disease human GLAST, BDNF, 2006
astrocyte GNDF
medium
CNS and PNS Peripheral and Peripheral Noggin, NGF Neural NCAM, Peripherin, No Brokhman
diseases central nervous sensory precursor TUJ-1, SNAIL, BRN3, TH, et al., 2008
system neurons neuron dHAND, SOX9 TRK-A
Macular Not applicable Retinal Noggin, Retinal RX, PAX6, RPE-65 No Lamba
retinal pigmented Dickkopf-1, progenitor LHX2, SIX3 et al., 2006
degeneration epithelium IGF-1

10
 Introduction

Neural Experimental Growth Progenitor Mature

Cell type Marker Transplantation Reference
disease model factor cell marker
Huntington’s — Striatal medium — — Islet1, — — Molero
disease spiny neuron DARPP-32, et al., 2009
specification mGluR1, NeuN
GABA neurons
Hirschsprung’s Engraftment and Enteric neurons LDN, SB, Enteric Sox10, TUJ-1, Yes Fattahi
disease functional rescue in CHIR, RA, nervous CD49d, PHOX2A, et al., 2016
HSCR mouse model FGF2, EGF, system EDNRB, TRKC, 5-HT,
GDNF, AA progenitors RET, HOXB2, GABA
HOXB5

References
Brokhman I, Gamarnik-Ziegler L, Pomp O, et al. (2008) Peripheral sensory neurons differentiate Lee DS, Yu K, Rho JY, et al. (2006) Cyclopamine treatment of human embryonic stem cells
from neural precursors derived from human embryonic stem cells. Differentiation 76:145–155. followed by culture in human astrocyte medium promotes differentiation into nestin- and
GFAP-expressing astrocytic lineage. Life Sci 80:154–159.
Fattahi F, Steinbeck JA, Kriks S, et al. (2016) Deriving human ENS lineages for cell therapy and
drug discovery in Hirschsprung disease. Nature 531:105–109. Li XJ, Du ZW, Zarnowska ED, et al. (2005) Specification of motoneurons from human embryonic
stem cells. Nat Biotechnol 23:215–221.
Giri M, Zhang M, and Lu Y (2016) Genes associated with Alzheimer’s disease: An overview and
current status. Clin Interv Aging 11:665–681. Molero AE, Gokhan S, Gonzalez S, et al. (2009) Impairment of developmental stem cell-
mediated striatal neurogenesis and pluripotency genes in a knock-in model of Huntington’s
Izrael M, Zhang P, Kaufman R, et al. (2007) Human oligodendrocytes derived from embryonic disease. Proc Natl Acad Sci U S A 106:21900–21905.
stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo. Mol
Cell Neurosci 34:310–323. Park CH, Minn YK, Lee JY, et al. (2005) In vitro and in vivo analyses of human embryonic stem
cell-derived dopamine neurons. J Neurochem 92:1265–1276.
Kang SM, Cho MS, Seo H, et al. (2007) Efficient induction of oligodendrocytes from human
embryonic stem cells. Stem Cells 25:419–424. Perrier AL, Tabar V, Barberi T, et al. (2004) Derivation of midbrain dopamine neurons from
human embryonic stem cells. Proc Natl Acad Sci U S A 101:12543–12548.
Keirstead HS, Nistor G, Bernal G, et al. (2005) Human embryonic stem cell-derived
oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord Roy NS, Cleren C, Singh SK, et al. (2006) Functional engraftment of human ES cell-derived
injury. J Neurosci 25:4694–4705. dopaminergic neurons enriched by coculture with telomerase-immortalized midbrain astrocytes.
Nat Med 212:1259–1268.
Kriks S, Shim J-W, Piao J, et al. (2011) Dopamine neurons derived from human ES cells
efficiently engraft in animal models of Parkinson’s disease. Nature 480:547–551. Trujillo CA, Schwindt TT, Martins AH, et al. (2009) Novel perspectives of neural stem cell
differentiation: From neurotransmitters to therapeutics. Cytometry A 75:38–53.
Lamba DA, Karl MO, Ware CB, and Reh TA. (2006) Efficient generation of retinal progenitor cells
from human embryonic stem cells. Proc Natl Acad Sci U S A 103:12769–12774. Yan Y, Yang D, Zarnowska ED, et al. (2005) Directed differentiation of dopaminergic neuronal
subtypes from human embryonic stem cells. Stem Cells 23:781–790.
Lee H, Shamy GA, Elkabetz Y, et al. (2007) Directed differentiation and transplantation of human
embryonic stem cell-derived motoneurons. Stem Cells 25:1931–1939.

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12
Neural cell culture and differentiation
3 Recovery, culture, and characterization of cortical and
hippocampal neurons

Summary

Primary neuronal cultures are indispensable in the fields of • Gibco™ Distilled Water (Cat. No. 15230162)
neurobiology and pharmacology. Many researchers prefer
to use freshly isolated neuronal cells, as they maintain their • Gibco™ DPBS, calcium, magnesium (Cat. No. 14040141)
functional viability. For convenience, however, an alternate • Gibco™ Neurobasal™ Plus Medium (Cat. No. A3582901)
route is to utilize cryopreserved neurons. The ability to
recover and culture primary neurons under serum-free • Gibco™ B-27™ Plus Supplement (50X) (Cat. No. A3582801)
conditions can reduce the variation that can arise from
• Gibco™ GlutaMAX™ Supplement (Cat. No. 35050061)
the isolation steps and facilitate tighter control of neuronal
studies. Some serum-free media and supplements allow
for the low-density culturing of neurons, which in turn Preparing media
enables the study of individual neurons and synapses. This
has not been possible using serum-supplemented media Complete Neurobasal Plus Medium
without a feeder layer of glial cells. In serum-supplemented Complete medium requires supplementation of Neurobasal
media, glial cells continue to multiply, necessitating the Plus Medium with B-27 Plus Supplement (50X) and
use of cytotoxic mitotic inhibitors. Serum also contains GlutaMAX Supplement. Complete medium is stable for
unknown and variable levels of growth factors, hormones, 2 weeks when stored in the dark at 2°C to 8°C.
vitamins, and proteins. This chapter details the recovery
and culture of cryopreserved primary neurons in serum- To prepare 100 mL of complete medium, aseptically mix
free media and supplements. the following components. For larger volumes, increase the
component amounts proportionally.
View this protocol online and order products at
thermofisher.com/neuroprotocol/neurons Component Final conc. Amount
Neurobasal Plus Medium 1X 98 mL
B-27 Plus Supplement (50X) 2% 2 mL
Required materials GlutaMAX Supplement 0.5 mM 250 μL
• Gibco™ Primary Rat Cortex Neurons, Sprague-Dawley
(Cat. No. A36511 or A36512)
Preparing matrix
• Gibco™ Primary Rat Hippocampus Neurons
(Cat. No. A1084101) Coating culture vessels with poly-D-lysine
1. Dilute the poly-D-lysine stock solution 1:40 in DPBS,
• Gibco™ Primary Mouse Cortical Neurons
calcium, magnesium (DPBS +/+), to prepare a 50 μg/mL
(Cat. No. A15585 or A15586)
working solution (e.g., 125 μL of poly-D-lysine stock
• Gibco™ Primary Mouse Hippocampal Neurons solution into 5 mL of DPBS +/+).
(Cat. No. A15587)
2. Coat the surface of the culture vessel with the
• Gibco™ Poly-D-Lysine (Cat. No. A3890401) working solution of poly-D-lysine (e.g., 50 μL/well for a
96-well plate).

13
Neural cell culture and differentiation

3. Incubate the vessel at room temperature for 1 hour. 1. Remove one 1-mL vial of frozen cells from
liquid nitrogen.
4. Remove the poly-D-lysine solution and rinse the
culture surface 3 times with sterile distilled water (e.g., 2. Thaw the vial in a 37°C water bath with gentle swirling.
100 μL/well for a 96-well plate).
3. Wipe down the vial with 70% ethanol and tap gently
Make sure to rinse the culture vessel thoroughly, as on a surface so that all of the medium collects at the
excess poly-D-lysine solution can be toxic to the cells. bottom of the vial.

5. Remove the distilled water and leave the coated culture 4. Open the vial in a laminar flow hood.
vessel uncovered in the laminar hood to dry.
5. Rinse a pipette tip with complete medium and very
The culture surface should be fully dry after 2 hours. gently transfer the cells from the vial to a pre-rinsed
15 mL tube.
Note: Coated vessels can be used immediately once
they are dry, or can be stored dry at 4°C. For storage at 6. Rinse the vial with 1 mL of complete medium (pre-
4°C, tightly wrap the vessels with Parafilm™ laboratory warmed to 37°C), and transfer the rinse to the 15 mL
film and use within one week of coating. tube containing the cells, at a rate of one drop per
second. Mix by gentle swirling after each drop.

Cell recovery 7. Slowly add 2 mL of complete medium to the tube (for a

total suspension volume of 4 mL).
Guidelines for recovery of cryopreserved neural cells
• Minimize the duration of exposure of neural cells to 8. Mix the suspension very gently with a P1000 pipette.
cryomedium at 37°C by thawing until only a small ice Avoid creating any air bubbles.
crystal remains. Avoid longer incubation at 37°C in
cryomedium solutions. 9. Using a pre-rinsed tip, add 10 μL of cell suspension to
a microcentrifuge tube containing 10 μL of 0.4% trypan
• Ensure that the complete medium is added to the neural blue. Mix the cells by gently tapping the tube. Determine
cells in cryomedium in a dropwise manner, to avoid the viable cell density using a hemocytometer or the
osmotic shock. Invitrogen™ Countess™ II Automated Cell Counter.

Recovering frozen neural cells 10. Plate to achieve the seeding density per well
Handle cells gently, because they are extremely fragile upon recommended for the B-27 Plus system (see Table 3-1
recovery from cryopreservation. It is important to rinse on the next page) in a poly-D-lysine–coated plate
pipette tips and vials with complete medium before using or a chambered slide. Bring the cell suspension
them for transferring cell suspensions, to prevent the cells volume to the desired volume per well by adding
from sticking to the plastic. Do not centrifuge cells upon complete medium.
recovery from cryopreservation.

14
 Neural cell culture and differentiation

Note: Use of a ROCK inhibitor (including Gibco™ Expected results

RevitaCell™ Supplement) is not recommended, as
it tends to rescue the glial population, which will Following the above protocol guidance and the
decrease the neuronal purity. When maximum B-27 Plus Neuronal Culture System protocol
cell viability is desired, add Gibco™ CultureOne™ (thermofisher.com/b27plus) will result in increased
Supplement (100X), which will suppress the neuronal survival, accelerated neurite outgrowth, and
proliferation of the contaminating glial population. improved neural network activity and maturation in
primary neurons (Figure 3-1 below and Figure 3-2 on the
11. Incubate the cells at 37°C in a humidified atmosphere of next page).
5% CO2 in air. A B
B-27 B-27 B-27 B-27
Plus Plus

12. Feed the cells on the next day and every third day
thereafter by aspirating half the medium from each well
and replacing it with fresh complete medium.

Table 3-1. Recommended seeding densities for

Seeding
Seeding
at 28,000
at 28,000
cells/cm
cells/cm
2 2
Seeding
Seeding
at 28,000
at 28,000
cells/cm
cells/cm
2 2

primary neurons.
Seeding density C Relative neuron count
250
Low Medium High*
Source Medium
(cells/cm 2) (cells/cm 2) (cells/drop)
(count/field, percent of B-27)

200
HuC/D-positive cells

B-27
≥40,000 ≥90,000 160,000
classic** 150
Rat
B-27 Plus
20,000 60,000 80,000
system 100

B-27
≥60,000 ≥100,000 120,000 50
classic**
Mouse
B-27 Plus 0
30,000 60,000 60,000 B-27 B-27 Plus
system
1–2 times Every 3–4 Every 2–3 Figure 3-1. Greater viability and quality of primary neurons with fewer
Feeding schedule†
weekly days days cells: mouse cortical neurons on culture day 14. Cells were plated at
* Multi-electrode array (MEA) application. 28,000 cells/cm2 and maintained in either (A) “classic” B-27 Supplement
** B-27 classic: B-27 Supplement with Neurobasal Medium. and Neurobasal Medium or (B) the B-27 Plus Neuronal Culture System
† Suggested half-volume change per feed. (B-27 Plus Supplement with Neurobasal Plus Medium). The number of
neurons (C) was determined by automated image capture and analysis
on the Thermo Scientific™ CellInsight™ CX5 High-Content Screening (HCS)
Platform with Thermo Scientific™ HCS Studio™ Cell Analysis Software.
Neurons were stained with MAP2 (green) and HuC/D (red) antibodies.
Nuclei were labeled with Invitrogen™ Hoechst 33342 (blue). For more
information about our antibodies and staining procedure, refer to the
Immunocytochemistry chapter (beginning on page 80).

15
Neural cell culture and differentiation

Electrophysiological activity Troubleshooting

12

10
For troubleshooting tips regarding cryopreservation and
Mean firing rate (Hz)

8 recovery of mature neurons, see below.

6 Problem Possible cause Solution
4 Low cell Neurons • Ensure that cells are
2
survival stressed during not left at 37°C for
recovery from extended periods
0
7 10 12 15 17 19 22 24 26 29 35 cryopreservation of time. Be certain
Days in culture to thaw neurons
until only a small ice
B-27 Supplement and B-27 Plus Neuronal Reagent from B-27 Supplement and
Neurobasal Medium Culture System another supplier Neurobasal Medium with serum

crystal remains.
Figure 3-2. The B-27 Plus system promotes better electrophysio­ • Ensure dropwise
logical activity than classic B-27 Supplement with serum. The B-27 addition of growth
Plus system (orange) promoted a higher degree of firing activity as the medium to the
network matured, as quantified via mean firing rate. Excitability of a neural
neurons to avoid
network was quantified by the mean firing rate, defined as the total number
of action potentials detected per second.
osmotic shock to
the cells.
• Limit the number
of vials thawed at
one time, as longer
exposure of cells
to cryopreservation
media can have a
negative impact on
post-thaw viability.
Non- Failure to prewet • Ensure prewetting
reproducible plastics of vials and plastics,
recovery including conical
tubes and pipette
tips, to minimize
loss of neurons
to the surface of
the plastics.

16
 Neural cell culture and differentiation

4 Thawing and culture of cryopreserved neurons

Summary Preparing media

The ability to cryopreserve primary neuronal cells is of great Complete Neurobasal medium
interest to many researchers. Flexibility in experimental Complete Neurobasal medium requires supplementation
workflows allows researchers to bank cells for use in of Neurobasal Medium with B-27 Supplement (50X), serum
basic research, translational medicine, and cell therapy free, and GlutaMAX Supplement. Complete medium is
applications. In this chapter we provide detailed protocols stable for 2 weeks when stored in the dark at 2°C to 8°C.
for recovery of cells and culture in complete Neurobasal
medium, which provides high post-thaw viability and To prepare 100 mL of complete Neurobasal medium,
recovery with superior neuronal quality. aseptically mix the following components. For larger
volumes, increase the component amounts proportionally.
View this protocol online and order products at
thermofisher.com/neuroprotocols Component Final conc. Amount
Neurobasal Medium 1X 98 mL
B-27 Supplement (50X), 2% 2 mL
Required materials serum free
GlutaMAX Supplement 0.5 mM 250 μL
Cells For primary hippocampus neuron cultures, complete Neurobasal medium requires additional
supplementation with 25 μM L-glutamate up to the fourth day of culture.
• Gibco™ Primary Rat Cortex Neurons (Cat. No. A1084001)

• Gibco™ Primary Rat Hippocampus Neurons

(Cat. No. A1084101)

• Gibco™ Primary Mouse Cortical Neurons (Cat. No. A15585)

• Gibco™ Primary Mouse Hippocampal Neurons

(Cat. No. A15587)

Media and reagents

• Gibco™ Neurobasal™ Medium (Cat. No. 21103049)

• Gibco™ B-27™ Supplement (50X), serum free

(Cat. No. 17504044)

• Gibco™ GlutaMAX™ Supplement (Cat. No. 35050061)

• Gibco™ Trypan Blue Solution, 0.4% (Cat. No. 15250061)

• Gibco Poly-D-Lysine (Cat. No. A3890401)

• 48-well plate or 8-chambered slides

• Gibco™ Distilled Water (Cat. No. 15230162)

• Gibco™ DPBS, calcium, magnesium (Cat. No. 14040141)

17
Neural cell culture and differentiation

Preparing matrix Recovery of neurons

Coating culture vessels with poly-D-lysine 1. Rinse a 50 mL conical culture tube with pre-warmed
1. Dilute the poly-D-lysine stock solution 1:40 in DPBS, (37°C) complete Neurobasal medium and leave it in the
calcium, magnesium (DPBS +/+), to prepare a 50 μg/mL laminar flow hood prior to thawing the cells.
working solution (e.g., 125 μL of poly-D-lysine stock
solution into 5 mL of DPBS +/+). 2. If removing a vial of cells from liquid nitrogen storage,
twist cap slightly to release pressure and then
2. Coat the surface of the culture vessel with the working retighten cap.
solution of poly-D-lysine (150 μL/cm2, e.g., 100 μL/well
for a 48-well plate). 3. Rapidly thaw (<2 minutes) the frozen vial by gently
swirling it in a 37°C water bath. Remove the vial from the
3. Incubate the culture vessel at room temperature water bath when only a tiny ice crystal is left (vial should
for 1 hour. still be cold to the touch).

4. Remove the poly-D-lysine solution and rinse the 4. Transfer the vial to the laminar flow hood and disinfect
culture surface 3 times with sterile distilled water (e.g., it with 70% isopropyl alcohol. Tap the vial gently on the
100 μL/well for a 96-well plate). surface of the hood so that the liquid settles down to the
bottom of the vial.
Make sure to rinse the culture vessel thoroughly, as
excess poly-D-lysine solution can be toxic to the cells. 5. Rinse a P1000 pipette tip with complete Neurobasal
medium and very gently transfer the cells to the pre-
5. Remove the distilled water and leave the coated culture rinsed 50 mL tube (from step 1).
vessel uncovered in the laminar hood to dry.
6. Rinse the vial with 1 mL of complete Neurobasal
The culture surface should be fully dry after 2 hours. medium (pre-warmed to 37°C) and add to the cells
in the 50 mL tube slowly, at the rate of one drop
Note: Coated vessels can be used immediately once per second. Mix the suspension by gentle swirling
they are dry, or can be stored dry at 4°C. For storage at after each addition.
4°C, tightly wrap the vessels with Parafilm™ laboratory
film and use within one week of coating. Note: Do not add the entire amount of medium to the
tube at once. This may lead to decreased cell viability
due to osmotic shock.

7. Slowly add 2 mL of complete Neurobasal medium to

the tube (for a total suspension volume of 4 mL). Mix the
suspension very gently with the P1000 pipette without
creating any air bubbles.

18
 Neural cell culture and differentiation

8. To a microcentrifuge tube containing 10 µL of Trypan Typical results

Blue Solution, 0.4%, add 10 µL of the cell suspension
using a pre-rinsed tip. Mix by gently tapping the tube. Thawed cortical neurons cultured in complete Neurobasal
Determine the viable cell density using a manual medium display extensive neurite outgrowth that continues
counting method (e.g., hemocytometer) . to increase as long as they are kept healthy in culture.
In contrast to non-cryopreserved neurons, post-thaw
Note: Do not centrifuge the cells, as they are extremely recovered neurons show dead cells due to post-thaw
fragile upon recovery from cryopreservation. toxicity, and typical cultures contain dead cells that stay
attached to the plate surface.
Note: It is important to rinse each pipette tip and
vial with complete Neurobasal medium before using
it for a cell suspension, to prevent the cells from
sticking to the plastic.

Culturing neurons

1. Plate ~5 x 104 live cells per well in a poly-D-lysine–

coated (4.5 µg/cm2) 48-well plate. Dilute the cell
suspension to 500 µL per well by adding complete
Neurobasal medium.

2. Incubate the cells at 36–38°C in a humidified

atmosphere of 5% CO2 in air.

3. After 4 to 24 hours of incubation, aspirate half of Figure 4-1. Phase-contrast image of primary rat cortical neurons
recovered and cultured in complete Neurobasal medium, at day 7.
the medium from each well and replace it with fresh
complete Neurobasal medium. Return the cells to
the incubator.

4. Feed the cells every third day by aspirating half

of the medium from each well and replacing it
with fresh medium.

Note: Do not expose neurons to ambient air

at any time.

19
Neural cell culture and differentiation

5 Culturing human fetal neural stem cells

Summary

Neural stem cells (NSCs) isolated from human fetuses Plasticware

are highly valuable resources in neuroscience because of • Thermo Scientific™ Nunclon™ Sphera™ Flasks
their ability to differentiate into neurons and glial cells. This (Cat. No. 174951 or 174952) (for suspension culture)
chapter describes methods for expanding human fetal
NSCs in cell culture and their subsequent characterization.
Preparing media
View this protocol online and order products at
thermofisher.com/neuroprotocol/hnsc Heparin stock
1. To prepare 6,000 U/mL heparin solution (1,000X), add
1.65 mL of distilled water to 10,000 U of heparin and mix
Required materials until dissolved.

Cells 2. After dissolving, filter-sterilize through a 0.22 µm filter,

• Gibco™ StemPro™ Neural Stem Cells (Cat. No. A15654) aliquot 50–100 µL into sterile tubes, and store at –20°C.

Media and reagents Ascorbic acid stock

• Gibco™ StemPro™ NSC SFM (Cat. No. A1050901: This kit 1. To prepare 100 mM ascorbic acid stock solution (500X),
contains KnockOut™ DMEM/F-12 Basal Medium stored add 17.27 mL of distilled water to 500 mg of ascorbic acid
at 4°C; StemPro™ NSC SFM (Neural) Supplement stored powder and mix until dissolved.
at –20°C to –5°C in the dark; and bFGF Recombinant
Human and EGF Recombinant Human proteins stored 2. After dissolving, filter-sterilize through a 0.22 µm filter,
at 4°C, desiccated.) aliquot 100–200 µL into sterile tubes, and store at –20°C.

• Gibco™ GlutaMAX™ Supplement (Cat. No. 35050061) bFGF and EGF stock
• Ascorbic Acid (MilliporeSigma, Cat. No. A8960) 1. Reconstitute bFGF and EGF with 0.1% BSA in DPBS,
calcium, magnesium (DPBS +/+, Cat. No. 14040133),
• Heparin (MilliporeSigma, Cat. No. H3149) or DPBS, no calcium, no magnesium (DPBS –/–,
Cat. No. 14190144), at a concentration of 20 μg/mL.
• Gibco™ Distilled Water (Cat. No. 15230162)

• Gibco™ DPBS, no calcium, no magnesium 2. Aliquot 50–100 µL into sterile tubes, and store at –20°C.
(Cat. No. 14190144)
Complete StemPro NSC SFM
• Gibco™ DPBS, calcium, magnesium (Cat. No. 14040133) To prepare 100 mL of complete neural stem cell culture
medium, mix the components shown in the table on
• Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent
the next page under sterile conditions. KnockOut
(Cat. No. A1110501)
DMEM/F-12, bFGF, EGF, and StemPro Neural Supplement
are components of the StemPro NSC SFM kit. Complete

20
 Neural cell culture and differentiation

medium is stable for 4 weeks when stored in the dark 5. Spin down the thawed cells by centrifugation at 300 x g
at 2°C to 8°C. To make larger volumes, increase the for 4 minutes. Aspirate and discard the supernatant.
component amounts proportionally.
6. Resuspend the cells in 1 mL complete StemPro
Component Final conc. Amount NSC SFM.
KnockOut DMEM/F-12 1X 96.5 mL
bFGF (prepared as 20 μg/mL stock) 20 ng/mL 100 μL 7. Determine the concentration of viable cells using your
EGF (prepared as 20 μg/mL stock) 20 ng/mL 100 μL preferred method.
StemPro Neural Supplement 2% 2 mL
GlutaMAX Supplement 2 mM 1 mL 8. Resuspend NSCs in pre-warmed complete StemPro
Heparin 6 units/mL 100 μL NSC SFM to a final concentration of 0.5 x 106 cells/cm2.
Ascorbic acid 200 μM 200 μL
9. Transfer 1 mL each of the cell suspensions to uncoated
T-25 flasks containing 6 mL of complete StemPro NSC
Culturing neural stem cells SFM. The total volume will be 7 mL for each T-25 flask
(~2 x 104 cells/cm2).
Fetal NSC populations can be expanded from frozen
stocks and grown as suspension cultures as plating on 10. Change the spent culture medium after 2 days and
matrix would trigger differentiation. The following procedure every 2–4 days thereafter.
provides instructions for thawing one vial of StemPro NSCs
containing 1 x 106 cells in two T-25 flasks (for a total culture 11. Culture the NSCs in suspension for up to 21 days,
area of 50 cm2). passaging them every 7 days.

Thawing frozen neural stem cells Change medium

1. Prepare 30 mL of complete StemPro NSC SFM and 1. Prepare 10 mL of complete StemPro NSC SFM per T-25
warm to 37°C. flask and pre-warm to 37°C.

2. Rapidly thaw (<2 minutes) the frozen vial by gently 2. From each culture flask, tilt and pipet the medium
swirling it in a 37°C water bath. Remove the vial from the containing the suspension cells into a corresponding
water bath when only a tiny ice crystal is left (vial should pre-labeled 15 mL centrifuge tube.
be still cold to touch).
3. Add 2 mL of complete StemPro NSC SFM (pre-warmed
3. Transfer the vial to the laminar flow hood and disinfect it to 37°C) to each flask, and place the flasks back into
with 70% ethanol. Allow the ethanol to evaporate before the incubator.
opening the vial.
4. Centrifuge the 15 mL tubes with the suspension cells at
4. Transfer thawed cells into a 15 mL tube and add 300 x g for 4 minutes and aspirate the supernatant to
complete StemPro NSC SFM, pre-warmed to 37°C, in a ~0.5 cm above the pellet surface without disturbing the
dropwise manner to a total volume of 5 mL. pellet. Discard the supernatant appropriately.

21
Neural cell culture and differentiation

5. Retrieve flasks from incubator (from step 3) and transfer 10 minutes. Swirl the tube at 5 and 8 minutes to ensure
them into the laminar flow hood. that the cells do not aggregate or settle at the bottom
of the tube.
6. Add 1 mL of warm medium into each tube (from
step 4) and dissociate the cells by gently pipetting up 6. Using a P1000 pipette, break up the neurospheres by
and down. Transfer the cell suspension back to the pipetting up and down 5 times. Place the tube back in
appropriate flask. the laminar flow hood for another 5–25 minutes.

7. Pipet another 4 mL of pre-warmed medium into each 7. Gently triturate neurospheres using a Pasteur pipette or
tube in a manner that washes the sides of the tube. P1000 pipettor to create a single-cell suspension.
Pipet the medium up and down the sides several
times. Transfer the cell suspension back to the 8. Neutralize the treatment by adding 4 mL of complete
appropriate flask. StemPro NSC SFM.

8. Mix the cell suspension evenly by gently moving flasks 9. Spin down the cells by centrifugation at 300 x g for
in a left-to-right and then forward-and-backward motion 3 minutes. Aspirate and discard the supernatant,
several times. and seed cells in fresh complete StemPro NSC
SFM in a suspension dish at a density of 2 x 104 to
9. Return flasks to the 37°C incubator with a humidified 5 x 104 cells/cm2.
atmosphere of 5% CO2 in air.
A B
Passaging neural stem cells
1. Transfer medium containing neurospheres into a 15 or
50 mL conical tube.

2. Leave the tube at room temperature and allow

the neurospheres to settle to the bottom of tube.
Alternatively, spin down the cells by centrifugation at
200 x g for 2 minutes.
Figure 5-1. Culture of neural stem cells. Phase-contrast image of cells
3. Aspirate the supernatant carefully, and leave the (A) 1 day after thawing of StemPro Neural Stem Cells and (B) 7 days after
thawing of StemPro Neural Stem Cells before harvest.
neurospheres in a minimum volume of medium.

4. Wash the neurospheres with 10 mL DPBS –/–, Cryopreserving neural stem cells
aspirate the DPBS supernatant carefully, and leave the 1. Harvest cells using method described above in
neurospheres in a minimum volume of DPBS. “Passaging neural stem cells”.

5. Add 1 mL of StemPro Accutase reagent to the 2. Resuspend the cells in complete StemPro NSC SFM at
neurospheres and incubate the tube at 37°C for a density of 2 x 106 cells/mL.

22
 Neural cell culture and differentiation

3. Prepare freezing medium (2X) consisting of 20% DMSO

and 80% complete StemPro NSC SFM.

Note: Freezing medium (2X) can be prepared on the

day of use and stored at 4°C until use.

4. Add a volume of freezing medium equal to the amount

of complete StemPro NSC SFM used to resuspend the
cells in a dropwise manner.

5. Prepare 1 mL aliquots (1 x 106 cells) in cryovials and

place the vials in an isopropyl alcohol chamber.

6. Put the isopropyl alcohol chamber at –80°C and transfer

the vials to liquid nitrogen storage the next day.

A Beta-3 tubulin GFAP B Beta-3 tubulin GFAP C O4 GFAP

Figure 5-2. Differentiation of StemPro NSCs. After 7 days of directed differentiation, (A) neurons,
(B) astrocytes, and (C) oligodendrocytes were labeled with phenotype markers of beta-3 tubulin
(neurons), GFAP (astrocytes), and O4 (oligodendrocytes).

23
Neural cell culture and differentiation

6 Culturing rat fetal neural stem cells

Summary

Rat neural stem cells (NSCs) serve as a well-established Special tools

model for investigating mammalian brain development, • Invitrogen™ Countess™ II Automated Cell Counter
disease processes, and for study of debilitating central (Cat. No. AMQAX1000) or hemocytometer
nervous system (CNS) disorders. This protocol describes
the in vitro expansion, passaging, and morphology of rat
fetal NSCs in adherent or neurosphere suspension cultures. Preparing media
View this protocol online and order products at Medium for expanding neural stem cells
thermofisher.com/neuroprotocol/ratnsc Complete StemPro NSC SFM consists of KnockOut
DMEM/F-12 with StemPro Neural Supplement, bFGF, EGF,
and GlutaMAX Supplement. Complete medium is stable for
Required materials 4 weeks when stored in the dark at 2°C to 8°C.

Cells To prepare 100 mL of complete StemPro NSC SFM:

• Gibco™ Rat Fetal Neural Stem Cells (Cat. No. N7744100)
or homogeneous cell preparation from 14−18 days post- 1. Reconstitute bFGF and EGF with 0.1% BSA solution (in
coitum rat brain tissue KnockOut DMEM/F-12) at a concentration of 100 μg/mL.
You will need 20 μL of each per 100 mL of complete
Media and reagents medium. Freeze unused portions in aliquots.
• Gibco™ DPBS, calcium, magnesium (Cat. No. 14040141)
2. Mix the following components under aseptic
• Gibco™ DPBS, no calcium, no magnesium
conditions. For larger volumes, increase the component
(Cat. No. 14190144)
amounts proportionally.
• Gibco™ StemPro™ NSC SFM (Cat. No. A1050901: This kit
Final
contains KnockOut™ DMEM/F-12 Basal Medium stored Component Amount
concentration
at 4°C; StemPro™ NSC SFM (Neural) Supplement stored
KnockOut DMEM/F-12 1X 97 mL
at –20°C to –5°C in the dark; and bFGF Recombinant
GlutaMAX Supplement 2 mM 1 mL
Human and EGF Recombinant Human proteins stored at
4°C, desiccated.) bFGF (prepared as 20 ng/mL 20 μL
100 μg/mL stock)
• Gibco™ StemPro Accutase™ Cell Dissociation Reagent EGF (prepared as 20 ng/mL 20 μL
(Cat. No. A1110501) 100 μg/mL stock)
StemPro Neural 2% 2 mL
• Gibco™ CELLstart™ CTS™ Substrate (Cat. No. A1014201) Supplement
You may observe a white precipitate when thawing StemPro Neural Supplement; this precipitate will
• Gibco™ Trypan Blue Solution, 0.4% (Cat. No. 15250061) disappear when the supplement is completely thawed or dissolved.
(included with the Invitrogen™ Countess™ II Automated Cell
Counter) or the Invitrogen™ LIVE/DEAD™ Cell Vitality Assay
Kit, C12 Resazurin/SYTOX™ Green (Cat. No. L34951)

24
 Neural cell culture and differentiation

Coating culture vessels with Expanding and passaging of rat NSCs

CELLstart CTS Substrate
Adherent cultures
For adherent cultures, prepare plates with CELLstart 1. Resuspend the rat fetal NSCs as follows:
CTS Substrate as described below.
• For freshly prepared rat fetal NSCs, after rinsing with
DPBS +/+, resuspend in warmed complete StemPro
1. Dilute CELLstart CTS Substrate 1:100 in DPBS, calcium,
NSC SFM at a density of 1 x 107 viable cells/mL.
magnesium (DPBS +/+) (e.g., 50 μL of CELLstart
CTS Substrate into 5 mL of DPBS). • For thawed rat fetal NSCs, after determining
the viable cell count, resuspend in warmed
Note: CELLstart CTS Substrate should not be frozen, complete StemPro NSC SFM at a cell density of
vortexed, or exposed to vigorous agitation due to 1 x 107 viable cells/mL.
potential gel formation.
2. Plate rat fetal NSCs onto culture vessels coated
2. Coat the surface of the culture vessel with the working with CELLstart CTS Substrate at a density of
solution of CELLstart CTS Substrate (14 mL for a T-75 5 x 104 cells/cm2. See the following table for
flask, 7 mL for a T-25 flask, 3.5 mL for a 60 mm dish, recommended seeding densities for common
2 mL for a 35 mm dish). culture vessels.

3. Incubate the culture vessel at 37°C in a humidified Volume No. of

Vessel size Growth area
of media cells
atmosphere of 5% CO2 for 1 hour.
96-well plate 0.32 cm2/well 0.1 mL 1.6 x 104
4. Remove the vessel from the incubator and store at 4°C 24-well plate 1.9 cm2/well 0.5 mL 1.0 x 105
until use. Remove all CELLstart CTS Substrate solution 12-well plate 3.8 cm2/well 1 mL 1.9 x 105
immediately before use, and fill the vessel with complete 35 mm dish 8 cm2/well 2 mL 4.0 x 105
StemPro NSC SFM. 6-well plate 9.6 cm2/well 2 mL 4.8 x 105
60 mm dish 19.5 cm 2
5 mL 9.8 x 105
Note: You may coat the plates in advance and store T-25 flask 25 cm2 5 mL 1.3 x 106
them at 4°C, wrapped tightly with Parafilm laboratory 100 mm dish 55 cm2 10 mL 2.8 x 106
film, for up to 2 weeks. Do not remove CELLstart CTS T-75 flask 75 cm2 15 mL 3.8 x 106
Substrate solution until just prior to using the coated
plates. Make sure the plates do not dry out. 3. Add the appropriate volume of cells to each
culture vessel and incubate at 37°C, 5% CO2, and
90% humidity.

4. Re-feed the rat fetal NSC cultures every 2−3 days with
fresh complete StemPro NSC SFM. The morphology
of rat fetal NSCs should exhibit short stellate-like
processes with uniform density (Figure 6-1 on the
next page).

25
Neural cell culture and differentiation

11. Resuspend the cell pellet in a minimal volume of pre-

warmed complete StemPro NSC SFM and remove a
sample for counting.

12. Determine the total number of cells and percent

viability using trypan blue stain or the LIVE/DEAD Cell
Vitality Assay Kit.

13. Add enough complete StemPro NSC SFM to the tube for
a final cell solution of 1 x 106 viable cells/mL. Incubate at
37°C, 5% CO2, and 90% humidity. Rat fetal NSC cultures
should not be maintained for more than 3 passages.

Important: If you are re-feeding rat fetal NSCs in a

Figure 6-1. Rat fetal NSCs at passage 3 in adherent culture using
complete StemPro NSC SFM. growth medium other than complete StemPro NSC SFM,
ensure that the medium is supplemented with 10 ng/mL
5. When cells reach 75–90% confluency (3–4 days bFGF to maintain the undifferentiated state of the rat
after seeding), the rat fetal NSC cultures are fetal NSCs.
ready to be passaged.
Neurosphere suspension cultures
6. Rinse the culture vessel once with DPBS –/–, then 1. Resuspend the rat fetal NSCs as follows:
remove the medium.
• For freshly prepared rat fetal NSCs, after rinsing with
DPBS –/–, resuspend in warmed complete StemPro
7. Add pre-warmed StemPro Accutase reagent and
NSC SFM at a cell density of 1 x 107 viable cells/mL.
let the cells detach from the culture surface (within
approximately 30 seconds). • For thawed rat fetal NSCs, after determining the viable
cell count, resuspend in warmed complete StemPro
8. After detachment, gently pipet the cells up and down NSC SFM at a cell density of 1 x 107 viable cells/mL.
to break the clumps into a uniform cell suspension
and add four volumes of complete StemPro NSC SFM 2. Plate the rat fetal NSCs onto uncoated or low-
to the culture vessel. attachment culture vessels at a density of
2 x 105 viable cells/cm2. See the table on the next page
9. Disperse the cells by pipetting over the culture surface for recommended seeding densities.
several times to generate a homogeneous cell solution.

10. Transfer the cells to a sterile centrifuge tube and

centrifuge at 300 x g for 4 minutes at room temperature.
Aspirate and discard the medium.

26
 Neural cell culture and differentiation

Volume of No. of 5. When the neurospheres reach a diameter of 200 μm or

Vessel size Growth area
media cells larger, the rat fetal NSCs are ready to be passaged.
96-well plate 0.32 cm2/well 0.1 mL 6.4 x 104
24-well plate 1.9 cm2/well 0.5 mL 3.8 x 105 6. Transfer the neurosphere suspension into a sterile
12-well plate 3.8 cm2/well 1 mL 7.6 x 105 centrifuge tube and let the neurospheres settle by
35 mm dish 8 cm2/well 2 mL 1.6 x 106 gravity or centrifuge at 200 x g for 2 minutes. Aspirate
6-well plate 9.6 cm /well2
2 mL 1.9 x 106 the supernatant carefully to leave the neurospheres in a
60 mm dish 19.5 cm2 5 mL 3.9 x 106 minimal volume of medium.
T-25 flask 25 cm2 5 mL 5.0 x 106
100 mm dish 55 cm2 10 mL 1.1 x 107 7. Rinse the neurospheres once with DPBS –/– and leave a
minimal volume of DPBS.
T-75 flask 75 cm 2
15 mL 1.5 x 10 7

8. Add 1 mL of pre-warmed StemPro Accutase reagent

3. Add the appropriate volume of cells to each culture to the neurospheres and incubate for 10 minutes
vessel and incubate at 37°C, 5% CO2, and 90% humidity. at room temperature.

4. Carefully re-feed the neurosphere suspension of rat fetal 9. After incubation, gently pipette the cells up and down to
NSCs every 2−3 days with fresh complete StemPro NSC get a single-cell suspension and add 4 mL of complete
SFM without removing any developing neurospheres. The StemPro NSC SFM to the tube.
morphology of the neurospheres should exhibit spherical
and transparent multicellular complexes (Figure 6-2). 10. Centrifuge at 300 x g for 4 minutes at room temperature,
carefully aspirate the supernatant, resuspend in a
minimal volume of pre-warmed complete StemPro
NSC SFM, and remove a sample for counting on a
hemocytometer or Countess II Automated Cell Counter.

11. Determine the total number of cells and percent viability.

12. Add enough complete StemPro NSC SFM to the tube

for a final cell solution of 1 x 107 viable cells/mL. Incubate
at 37°C, 5% CO2, and 90% humidity. Neurosphere
suspension cultures should not be maintained for more
than 3 passages.

Important: If you are re-feeding rat fetal NSCs in a

Figure 6-2. Rat fetal NSCs at passage 3 in neurosphere culture using growth medium other than complete StemPro NSC
complete StemPro NSC SFM. SFM, ensure that the medium is supplemented with
10 ng/mL bFGF to maintain the undifferentiated state of
the rat fetal NSCs.

27
Neural cell culture and differentiation

7 Differentiating glial precursor cells into astrocytes

and oligodendrocytes

Summary

Glial precursor cells (GPCs), also known as glial restricted • T3 (3,3�,5-triiodo-L-thyronine) (MilliporeSigma,
progenitors (GRP) or oligodendrocyte progenitor cells Cat. No. T6397)
(OPCs), are cells that have the potential to differentiate
into oligodendrocytes or astrocytes. The GPC population • N-acetyl-L-cysteine (MilliporeSigma, Cat. No. A8199)
is derived from tissue or is generated from pluripotent • Gibco™ BDNF Recombinant Human Protein
cells by differentiation, which is induced by exogenously (Cat. No. PHC7074)
applied factors. Here we describe a culture system
that can be adjusted to favor differentiation into either • Gibco™ CNTF Recombinant Human Protein
astrocytes or oligodendrocytes. (Cat. No. PHC7015)

• Gibco™ GlutaMAX™ Supplement (Cat. No. 35050061)

View this protocol online and order products at
thermofisher.com/neuroprotocol/gpc • Gibco™ Distilled Water (Cat. No. 15230162)

• Gibco™ DPBS, no calcium, no magnesium

Required materials (Cat. No. 14190144)

• Gibco™ DPBS, calcium, magnesium (Cat. No. 14040133)

Cells
• Gibco™ Rat Glial Precursor Cells (Cat. No. N7746100) • Gibco™ Geltrex™ LDEV-Free Reduced Growth Factor
Basement Membrane Matrix (Cat. No. A1413201)
Media and reagents
• Gibco™ StemPro™ NSC SFM (Cat. No. A1050901: This kit • Gibco™ Laminin Mouse Protein, Natural
contains KnockOut™ DMEM/F-12 Basal Medium stored at (Cat. No. 23017015)
4°C; StemPro NSC SFM (Neural) Supplement stored at
™

• Poly-L-Ornithine Solution (MilliporeSigma,

–20°C to –5°C in the dark; and bFGF Recombinant Human
and EGF Recombinant Human proteins stored at 4°C, Cat. No. P4957)
desiccated.)

• Gibco™ Neurobasal™ Plus Medium (Cat. No. A3582901)

Preparing media

• Gibco™ B-27™ Plus Supplement (50X), serum free PDGF-AA stock

(Cat. No. A3582801) 1. To prepare 10 µg/mL PDGF-AA stock solution (500X),
centrifuge the 10 µg vial briefly before aseptically adding
• Gibco™ Astrocyte Medium (Cat. No. A1261301: This
1 mL sterile 4 mM HCl containing 0.1% BSA.
kit contains N-2 Supplement (100X) stored at –20°C;
Dulbecco’s Modified Eagle Medium (DMEM) (1X) stored
2. Aliquot 50–100 µL into sterile tubes, and store at –20°C.
at 2°C to 8°C; One Shot™ Fetal Bovine Serum, Certified,
stored at –20°C in the dark.)
T3 stock
• Gibco™ PDGF-AA Recombinant Human Protein 1. To prepare 30 µg/mL T3 stock solution (1,000X),
(Cat. No. PHG0035) add 1 mL of 1 N NaOH to 1 mg of T3 powder, and

28
 Neural cell culture and differentiation

then dilute further with 32.3 mL of DPBS, calcium, To prepare 100 mL of complete medium:
magnesium (DPBS +/+), and mix until dissolved.
1. Reconstitute bFGF and EGF with 0.1% BSA solution (in
2. After dissolving, filter-sterilize through a 0.22 µm filter, KnockOut DMEM/F-12) at a concentration of 100 μg/mL.
aliquot 50–100 µL into sterile tubes, and store at –20°C. You will need 20 μL of each per 100 mL of complete
medium. Freeze unused portions in aliquots.
N-acetyl-L-cysteine stock
1. To prepare 5 mg/mL N-acetyl-L-cysteine stock, dissolve 2. Mix the following components under aseptic
50 mg N-acetyl-L-cysteine in 10 mL Neurobasal Plus conditions. For larger volumes, increase the component
Medium by vortexing. amounts proportionally.

2. Add 1 N NaOH dropwise to neutralize pH; requires Component Final conc. Amount
approximately 250 µL until medium returns to original KnockOut DMEM/F-12 1X 97 mL
red color. GlutaMAX Supplement 2 mM 1 mL
bFGF (prepared as 20 ng/mL 20 μL
3. Filter-sterilize through a 0.22 µm filter, aliquot 50–100 µL 100 μg/mL stock)
into sterile tubes, and store at –20°C. EGF (prepared as 20 ng/mL 20 μL
100 μg/mL stock)
BDNF stock StemPro Neural 2% 2 mL
1. To prepare 10 µg/mL BDNF stock solution (5,000X), Supplement
aseptically add 1 mL of DPBS +/+ supplemented You may observe a white precipitate when thawing StemPro Neural Supplement; swirl thawed
supplement briefly in a 37°C bath until precipitate dissolves.
with 0.1% BSA to 10 µg of BDNF protein, and mix
until dissolved.
3. Add 20 ng/mL PDGF-AA to prewarmed complete
2. Aliquot 10–20 µL into sterile tubes, and store at –20°C. StemPro NSC SFM immediately prior to use.

CNTF stock Oligodendrocyte differentiation medium

1. To prepare 10 µg/mL CNTF stock solution (5,000X), Oligodendrocyte differentiation medium uses Neurobasal
aseptically add 2 mL of DPBS +/+ supplemented Plus Medium supplemented with B-27 Plus Supplement
with 0.1% BSA to 20 µg of CNTF protein, and mix (50X), N-acetyl-L-cysteine, T3, BDNF, and CNTF. Complete
until dissolved. medium is stable for 2 weeks when stored in the dark at
2°C to 8°C.
2. Aliquot 10–20 µL into sterile tubes, and store at –20°C.
To prepare 100 mL of oligodendrocyte differentiation
Complete StemPro NSC SFM medium, mix the components shown in the table on the
Complete StemPro NSC SFM consists of KnockOut next page under aseptic conditions. For larger volumes,
DMEM/F-12 with StemPro Neural Supplement, GlutaMAX increase the component amounts proportionally.
Supplement, bFGF, and EGF. Complete medium is stable
for 2 weeks when stored in the dark at 2°C to 8°C.

29
Neural cell culture and differentiation

Component Final conc. Amount 6. Incubate the culture vessel at 37°C in a humidified
Neurobasal Plus Medium 1X 98 mL atmosphere of 5% CO2 for at least 1 hour. Store it at
B-27 Plus Supplement 2% 2 mL 4°C until use.
N-acetyl-L-cysteine 5 μg/mL 100 μL
T3 30 ng/mL 100 μL Note: You may coat the plates in advance and store
BDNF 2 ng/mL 20 µL them at 2°C to 8°C, wrapped tightly with Parafilm
CNTF 2 ng/mL 20 µL laboratory film, for up to 4 weeks.

Matrix for astrocyte differentiation

Astrocyte differentiation medium 1. Thaw a bottle of Geltrex Basement Membrane Matrix at
Astrocyte differentiation medium uses DMEM 2°C to 8°C.
supplemented with N-2 Supplement and FBS. Complete
medium is stable for 2 weeks when stored in the dark at 2. Dilute Geltrex matrix 1:100 in cold DMEM and coat the
2°C to 8°C. bottom of each culture vessel by adding 2 mL of diluted
Geltrex matrix to a 35 mm dish (0.5 mL for 4-well plate
Component Final conc. Amount or slide, 0.25 mL for 8-well slide).
DMEM 1X 97 mL
N-2 Supplement 1% 1 mL 3. Incubate the culture vessel at 36°C to 38°C for 1 hour.
FBS 1% 2 mL
Note: Dishes coated with Geltrex matrix can be used
immediately or stored at 2°C to 8°C for up to one
Preparing matrix week, sealed with Parafilm laboratory film. Do not allow
dishes to dry out. Warm stored Geltrex matrix plates to
Matrix for oligodendrocyte differentiation room temperature for 1 hour prior to use.
1. Add 2 mL of 100 μg/mL poly-L-ornithine solution to a
35 mm dish (0.5 mL for 4-well plate or slide, 0.25 mL for Differentiation to oligodendrocytes
8-well slide). 1. Plate glial precursor cells on a poly-L-ornithine– and
laminin-coated plate in complete StemPro NSC SFM
2. Incubate the culture vessel at 37°C in a humidified supplemented with 20 ng/mL PDGF-AA at a density of
atmosphere of 5% CO2 for at least 1 hour. 2.5 x 104 to 5 x 104 cells/cm2.

3. Rinse the culture vessel 3 times with distilled water. 2. On the next day, change the medium to
oligodendrocyte differentiation medium.
4. Prepare a 1:100 dilution of laminin in distilled water for a
final concentration of 10 μg/mL. 3. Perform a half-medium change every 2–3 days
thereafter. Differentiated oligodendrocytes are typically
5. Add 2 mL of the 10 μg/mL laminin solution to a 35 mm observed on day 3.
dish (0.5 mL for 4-well plate or slide, 0.25 mL for
8-well slide).

30
 Neural cell culture and differentiation

Differentiation to astrocytes
1. Plate glial precursor cells on a Geltrex matrix–
coated plate in complete StemPro NSC SFM
supplemented with 20 ng/mL PDGF-AA at a density
of 2.5 x 104 cells/cm2.

2. On the next day, change the medium to astrocyte

differentiation medium.

3. Change the medium every 3–4 days. Differentiated

astrocytes are typically observed on days 5–7.

31
Neural cell culture and differentiation

8 Xeno-free culture of neural stem cells

Summary Required materials

Neural stem cells (NSCs) derived from human pluripotent Cells

stem cells (hPSCs) help provide understanding for • Neural stem cells
human neurogenesis and have potential for cell therapy
applications related to Parkinson’s disease, spinal cord Media and reagents
injuries, and other neurological diseases. Standard • Gibco™ CELLstart™ CTS™ Substrate (Cat. No. A1014201)
methods of culturing NSCs raise concerns about pathogen
• Gibco™ CTS™ KnockOut™ DMEM/F-12 (Cat. No. A1370801)
cross-transfer from non-human sources or contamination
with non-neural cells, limiting the efficiency and specificity • Gibco™ CTS™ N-2 Supplement (Cat. No. A1370701)
of the differentiation protocols. These concerns have led
to the development of xeno-free conditions for maintaining • Gibco™ CTS™ B-27™ Supplement, XenoFree
and expanding NSCs, which are described in this protocol. (Cat. No. A1486701)
Gibco™ Cell Therapy Systems (CTS™) products help
• Gibco™ CTS™ FGF-Basic Full Length Recombinant Human
minimize the risk of contamination and variability, and meet
Protein (Cat. No. CTP0261)
your regulatory and quality requirements.
• Gibco™ EGF Recombinant Human Protein
View this protocol online and order products at (Cat. No. PHG0311)
thermofisher.com/neuroprotocols
• Gibco™ CTS™ GlutaMAX™-I Supplement
Learn more about CTS products at (Cat. No. A1286001)
thermofisher.com/cts
• Gibco™ CTS™ TrypLE™ Select Enzyme (Cat. No. A1285901)

• Gibco™ CTS™ Dulbecco’s Phosphate-Buffered Saline

(DPBS), calcium, magnesium (Cat. No. A1285801)

• Gibco™ CTS™ Dulbecco’s Phosphate-Buffered Saline

(DPBS), without calcium chloride, without magnesium
chloride (Cat. No. A1285601)

Special tools
• 15 mL conical tube

• Tissue culture plates and flasks

• Microcentrifuge

32
 Neural cell culture and differentiation

Preparing culture vessels and media

CELLstart CTS Substrate–coated vessels Culture medium

Prepare culture dishes or flasks with CELLstart Prepare 50 mL of culture medium by aseptically mixing the
CTS Substrate as described below. components in the table below.

1. Dilute CELLstart CTS Substrate 1:100 in CTS DPBS Component Amount

calcium, magnesium (CTS DPBS +/+), (e.g., 50 μL of CTS KnockOut DMEM/F-12 48.5 mL
CELLstart CTS Substrate into 5 mL of CTS DPBS +/+). CTS GlutaMAX-I Supplement 0.5 mL
CTS B-27 Supplement, XenoFree 1 mL
Note: CELLstart CTS Substrate should not be frozen, CTS N-2 Supplement 0.5 mL
vortexed, or exposed to vigorous agitation due to CTS FGF-Basic 20 ng/mL
potential gel formation. EGF 20 ng/mL

2. Coat the surface of the culture vessel with the working

solution of CELLstart CTS Substrate (2.5 mL for a T-25 The growth factors can be added immediately before use.
flask or 60 mm dish, 1.5 mL for a 35 mm dish). After the culture medium has been supplemented with
growth factors, aliquot the amount needed for the day
3. Incubate the culture vessel at 37°C in a humidified and store the remaining medium at 4°C. Complete culture
atmosphere of 5% CO2 for 1 hour. medium is stable for 2 weeks if properly stored at 4°C.

4. Use the dish immediately after incubation. Aspirate

the CELLstart CTS Substrate solution immediately Methods
before use.
Thawing and seeding NSCs
Note: Prepare a freshly coated culture vessel each 1. Remove a vial of cells from liquid nitrogen and quickly
time before plating cells. There is no need to rinse the thaw the vial in a 37°C water bath, being careful not to
culture vessel before use. immerse the vial above the level of the cap.

2. When just a small crystal of ice remains, remove vial

from water bath (<2 minutes) and sterilize the outside of
the vial with 95% ethanol. Allow the ethanol to evaporate
before opening the vial in a laminar flow hood.

3. Gently pipet the cell suspension up and down once, and

place it into a 15 mL centrifuge tube.

4. Add 10 mL of warm culture medium to the tube

dropwise to reduce osmotic shock.

33
Neural cell culture and differentiation

5. Centrifuge the cell suspension at 200 x g for 5 minutes. 7. Add 4 mL of culture medium to neutralize the CTS
TrypLE Select Enzyme activity and pipet up and down
6. Remove the supernatant, resuspend the pellet in 5 mL 2–3 times to get a uniform cell suspension. Check the
of culture medium, and determine the total number of cells under a microscope.
cells and percent viability.
8. Transfer the cell suspension to a 15 mL centrifuge tube.
7. Seed the cells at a concentration of 1 x 105 cells/cm2
onto a dish or flask that has been treated with CELLstart 9. Centrifuge the cells at 200 x g for 5 minutes.
CTS solution. (Aspirate the CELLstart CTS solution
immediately before using the dish or flask.) 10. Aspirate the supernatant and resuspend the cells in
10 mL of culture medium.
8. Incubate at 36°C to 38°C in a humidified atmosphere
(90%) of 5% CO2 in air. 11. Determine the total number of cells and percent viability
using your method of choice. Seed the cells at a
Culture and propagation concentration of 1 x 105 cells/cm2 onto a dish or flask
1. Twenty-four hours after seeding the cells, replace the that has been treated with CELLstart CTS solution.
culture medium.
12. Incubate the flasks at 37°C in a humidified atmosphere
2. Replace the spent medium every other day with an (90%) of 5% CO2 in air.
equal volume of fresh culture medium.
13. Grow the cells until 90% confluent, changing the culture
Note: If the medium turns yellow, change the medium once after 12 hours and every 2 days thereafter.
medium daily. Yellow medium will affect the
NSC proliferation rate. A B

3. After 4–7 days the culture will be 90% confluent and

ready for passaging.

4. To passage the culture, prepare a 0.5X solution of CTS

TrypLE Select Enzyme in CTS DPBS, without calcium
chloride, without magnesium chloride (CTS DPBS –/–).

5. Aspirate the culture medium and wash the cells with Figure 8-1. Phase-contrast microphotograph showing (A) NSCs
cultured in xeno-free medium 24 hours post-thaw and (B) semi-
CTS DPBS –/–. confluent NSCs cultured in xeno-free medium for 3 days.

6. Add 0.5X CTS TrypLE Select Enzyme to dissociate

the cells, and incubate for 2 minutes at 37°C or until
the cells start to round up and separate from the
culture surface.

34
 Neural cell culture and differentiation

9 Creation of neural stem cells from human pluripotent

stem cells

Summary

The derivation of neural stem cells (NSCs) is the first step in in the dark for up to 1 year. The thawed Y-27632 solution
producing various neural cell types from human pluripotent can be kept at 2°C to 8°C for up to 2 weeks.
stem cells (hPSCs). We have developed an efficient neural
• Gibco™ DPBS, no calcium, no magnesium
induction medium that can convert hPSCs into NSCs
(Cat. No. 14190250)
in one week without the need for the tedious and time-
consuming process of embryoid body formation. Neural • Gibco™ Essential 8™ Medium (Cat. No. A1517001)
induction medium works with hPSCs cultured either in
• Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent
feeder-containing or feeder-free conditions.
(Cat. No. A1110501): Thawed Accutase reagent can be
kept at 2°C to 8°C for up to 2 weeks.
View this protocol online and order products at
thermofisher.com/neuroprotocols • Dimethyl sulfoxide, Hybri-Max™ grade (DMSO)
(MilliporeSigma, Cat. No. D2650)

Required materials Special tools

• Thermo Scientific™ Nunc™ Cell Scraper (Cat. No. 179693
Media and reagents or 179707)
• Gibco™ PSC Neural Induction Medium
• 100 µm strainer
(Cat. No. A1647801): This kit contains 1X Neurobasal™
Medium stored at 4°C and 50X Neural Induction • Thermo Scientific™ Nalgene™ General Long-Term Storage
Supplement stored at –20°C to –5°C in the dark. After Cryogenic Tubes (Cat. No. 5000-0012 or 5000-1012)
thawing of Neural Induction Supplement, aliquot 0.5 to
1 mL into sterile tubes and store at –20°C to –5°C in the • Thermo Scientific™ Mr. Frosty™ Freezing Container
dark. Avoid frequent freezing and thawing. (Cat. No. 5100-0001)

• Gibco™ Advanced DMEM/F-12 (Cat. No. 12634010) • Microscope marker, such as Nikon™ microscopy object
marker (Nikon Instruments Inc., Cat. No. MBW10020)
• Gibco™ Geltrex™ LDEV-Free, hESC-Qualified, with a Nikon™ microscopy C-OA 15 mm objective adapter
Reduced Growth Factor Basement Membrane Matrix (Nikon Instruments Inc., Cat. No. MXA20750)
(Cat. No. A1413301): Thaw Geltrex matrix at 4°C
overnight before using. The thawed Geltrex matrix can be
kept at 2°C to 8°C for up to 2 weeks.
Preparing media
• Gibco™ Distilled Water (Cat. No. 15230162)
Neural induction medium
• ROCK Inhibitor Y-27632 (MilliporeSigma, Cat. No.
To prepare 100 mL of neural induction medium, mix the
Y0503): Dissolve 1 mg of Y-27632 in 0.625 mL distilled
following components under aseptic conditions. For larger
water to make 5 mM stock solution. Sterilize the stock
solution by filtering through a 0.22 µm filter. Aliquot volumes, increase the component amounts proportionally.
20–50 µL into sterile tubes and store at –20°C to –5°C

35
Neural cell culture and differentiation

Component Final conc. Amount 2. When hPSCs reach ~70–80% confluency, split hPSCs
Neurobasal Medium 1X 98 mL into 6-well culture plates to reach 15–25% confluency at
Neural Induction Supplement 1X 2 mL day 1 of hPSC splitting.

The complete neural induction medium can be stored at 3. At day 1 of hPSC splitting, aspirate spent medium to
2°C to 8°C for up to 2 weeks. When feeding cells, warm remove non-attached cells and add 2.5 mL pre-warmed
up the appropriate amount of neural induction medium in a neural induction medium into each well of 6-well plate.
37°C water bath for 5–10 minutes before feeding. Return plates to an incubator.

Neural expansion medium 4. The morphology of cell colonies should be uniform with
To prepare 100 mL of neural expansion medium, mix the a smooth edge after 2 days of culture in neural induction
following components under aseptic conditions. For larger medium (Figure 9-1, panel A). Due to differentiated or
volumes, increase the component amounts proportionally. partially differentiated hPSCs before neural induction,
colonies of non-neural differentiation can be observed
Component Final conc. Amount (Figure 9-1, panels B–F). Mark all colonies showing the
Neurobasal Medium 0.5X 49 mL morphology of non-neural differentiation on the bottom
Advanced DMEM/F-12 0.5X 49 mL of the plate by using a microscope marker.
Neural Induction Supplement 1X 2 mL

The complete neural expansion medium can be stored at

2°C to 8°C for up to 2 weeks. When feeding cells, warm
up the appropriate amount of neural induction medium in a
37°C water bath for 5–10 minutes before feeding.

Neural preservation medium

To prepare 10 mL of neural preservation medium, mix the
following components under aseptic conditions. For larger
volumes, increase the component amounts proportionally. Figure 9-1. Morphology of neural and non-neural differentiated cells
at day 2 of neural induction. (A) Normal morphology of cells. (B–F)
Representative images of non-neural differentiated cells (indicated by
Component Final conc. Amount
arrows) due to the use of poor-quality starter hPSCs.
Neural expansion medium 80% 8 mL
DMSO 20% 2 mL 5. Aspirate spent medium from each well. Remove all
colonies of non-neural differentiation by pointing a
Pasteur glass pipette to marked colonies to aspirate
Neural induction cells off. Add 2.5 mL pre-warmed neural induction
medium per well and return plates to an incubator.
1. Maintain the high quality of hPSCs including hESCs
and hiPSCs by culturing in feeder-free media such as 6. At day 4 of neural induction, cells should reach
Essential 8 Medium or complete StemPro hESC SFM, or confluency. Mark all colonies of non-neural differentiation
on mouse embryonic fibroblasts (MEFs). by using a microscope marker.

36
 Neural cell culture and differentiation

7. Aspirate spent medium from each well. Remove all 9. Triturate cell suspension 3 times with a 5 or 10 mL
colonies of non-neural differentiation by pointing a pipette to break cell clumps.
Pasteur glass pipette to marked colonies to aspirate
cells off. Add 5 mL pre-warmed neural induction 10. Pass cell suspension through a 100 µm strainer and
medium per well, and return plates into an incubator. centrifuge cells at 300 x g for 4 minutes.

8. Feed cells every day with 5 mL neural induction medium 11. Aspirate supernatant, resuspend cells with appropriate
per well until day 7 of neural induction. amount of pre-warmed neural expansion medium
(e.g., 1 mL for all cells from 1 well of a 6-well plate).

NSC expansion 12. Determine cell concentration using preferred method.

1. At day 7 of neural induction, NSCs (P0) are ready 13. Dilute cell suspension to 4 x 105 cells/mL with pre-
to be harvested. warmed neural expansion medium.

2. Dilute thawed Geltrex matrix in Neurobasal Medium 14. Add ROCK inhibitor Y-27632 solution into cell
(1:100). Add appropriate amount of Geltrex solution into suspension to reach final concentration of 5 µM.
each culture vessel to cover the surface (e.g., 0.5–1 mL
for each well of a 6-well culture plate) and incubate at 15. Aspirate Geltrex solution from coated vessels and
least 1 hour at 37°C. add appropriate amount of diluted cell suspension
into each culture vessel to plate cells at a density of
3. Aspirate the spent medium with a Pasteur glass pipette 1 x 105 cells/cm2.
and rinse cells with DPBS –/– once.
16. Place vessels gently in an incubator and move culture
4. Gently add 1 mL pre-warmed StemPro Accutase Cell vessels in several quick back-and-forth and side-to-side
Dissociation Reagent to each well of 6-well plate. motions to disperse cells across the surface of vessels.

5. Incubate for 5–8 minutes at 37°C until most of cells are 17. Change neural expansion medium at day 1 of cell
detached from the surface of culture vessels. plating to eliminate Y-27632. Change neural expansion
medium every other day thereafter.
6. Use a cell scraper to detach the cells from the surface
of the plates. 18. Usually, NSCs reach confluency at day 4–5 after plating.
When NSCs reach confluency, NSCs can be further
7. Transfer cell clumps using a pipette, and place cells into expanded in neural expansion medium. For the first
a 15 or 50 mL conical tube. 3 to 4 passages, overnight treatment with 5 µM Y-27632
at the day of NSC plating is necessary to prevent
8. Add 1 mL DPBS –/– to each well of a 6-well plate to cell death.
collect residual cells, and transfer cell suspension to
the conical tube.

37
Neural cell culture and differentiation

Cryopreservation of NSCs 6. Quickly remove the sticker or copy the information

written on the vial in your notebook. The writing may
1. After determining cell concentration at step 12 in the come off the vial after spraying the outside of the vial
NSC expansion section above, dilute cell suspension to with 70% ethanol.
2 x 106 to 4 x 106 cells/mL with neural expansion medium.
7. Spray the outside of the vial with 70% ethanol and place
2. Add an equivalent volume of neural preservation in a laminar flow hood. Pipette cells gently into a sterile
medium dropwise. 15 mL conical tube using a 1 mL pipette.

3. Allocate 1 mL of cell suspension into each 8. Add 1 mL DPBS –/– into the vial to collect residual cells.
cryotube and freeze cells at –80°C overnight in
Mr. Frosty Freezing Containers. 9. Use a pipette to remove DPBS–cell mixture from the vial
and add it to the 15 mL conical tube dropwise. While
4. Transfer cells into liquid nitrogen tank on the next day adding, gently move the tube back and forth to mix
for long-term storage. NSCs. This reduces osmotic shock to cells.

10. Centrifuge at 300 x g for 5 minutes and aspirate

Recovery of cryopreserved NSCs the supernatant.

1. Coat culture vessels with Geltrex solution for at least 11. Resuspend the cell pellet in DPBS –/–, centrifuge at
1 hour before thawing NSCs by following the procedures 300 x g for 5 minutes, and aspirate the supernatant.
of step 2 in the NSC expansion section above.
12. Resuspend cell pellet in appropriate amount of pre-
2. Wear eye protection, as cryotubes stored in the liquid warmed neural expansion medium (e.g., 1 mL for all
phase of liquid nitrogen may accidentally explode NSCs from 1 vial) and determine cell concentration
when warmed. using preferred method.

3. Wear ultralow-temperature cryogloves. Remove 13. Dilute cell suspension to 4 x 105 cells/mL with pre-
cryotubes of NSCs from the liquid nitrogen storage tank warmed neural expansion medium. If the passage
using metal forceps. number of the NSCs is less than 4, add ROCK inhibitor
Y-27632 solution into the cell suspension to reach final
4. Immerse the vial in a 37°C water bath without concentration of 5 µM.
submerging the cap. Swirl the vial gently.
14. Aspirate Geltrex solution from coated vessels and
5. When only an ice crystal remains, remove the vial from add an appropriate amount of diluted cell suspension
the water bath. into each culture vessel to plate cells at the density of
1 x 105 cells/cm2.

38
 Neural cell culture and differentiation

15. Place vessels gently in an incubator and move culture

vessels in several quick back-and-forth and side-to-side
motions to disperse cells across the surface of vessels.

16. Change neural expansion medium every other day

thereafter until NSCs reach confluency for splitting.

39
Neural cell culture and differentiation

10 Differentiating human pluripotent stem cell–derived

neural stem cells into neurons

Summary

Neural stem cells (NSC) derived from human pluripotent • Gibco™ DPBS, no calcium, no magnesium
stem cells (hPSCs) are self-renewing multipotent stem cells (Cat. No. 14190144)
that can be further differentiated into downstream lineages
such as neurons and glial cells. The protocols described • ROCK Inhibitor Y-27632 (MilliporeSigma, Cat. No. Y0503):
herein are primarily optimized with NSCs derived from Dissolve 1 mg of Y-27632 in 0.31 mL distilled water to
hPSCs using Gibco™ PSC Neural Induction Medium. make 10 mM stock solution. Sterilize the stock solution
by filtering through a 0.22 µm filter. Aliquot 20–50 µL into
View this protocol online and order products at sterile tubes and store at –20°C to –5°C in the dark for
thermofisher.com/neuroprotocols up to 1 year. The thawed Y-27632 solution can be kept at
2°C to 8°C for up to 2 weeks.

• L-ascorbic acid 2-phosphate sesquimagnesium salt

Required materials hydrate (MilliporeSigma, Cat. No. A8960): Dissolve 1 g
of ascorbic acid 2-phosphate sesquimagnesium salt
Cells hydrate in 17.3 mL distilled water, filter-sterilize through a
• NSCs derived by PSC Neural Induction Medium 0.22-μm filter, aliquot 100–200 μL into sterile tubes, and
store at –20°C to –5°C in the dark for up to 6 months.
Media and reagents
• Gibco™ Neurobasal™ Medium (Cat. No. 21103049) Equipment
• 37°C humidified cell culture incubator with 5% CO2
• Gibco™ Neurobasal™ Plus Medium (Cat. No. A3582901)
• Centrifuge
• Gibco™ B-27™ Supplement (50X), serum free
(Cat. No. 17504044) • 37°C water bath

• Gibco™ B-27™ Plus Supplement (50X) (Cat. No. A3582801) Consumables

• Corning™ BioCoat™ Poly-D-Lysine Multiwell Plates: 96-well
• Gibco™ CultureOne™ Supplement (100X)
(Fisher Scientific, Cat. No. 08-774-255), 48-well (Fisher
(Cat. No. A3320201)
Scientific, Cat. No. 08-774-288), 24-well (Fisher Scientific,
• Gibco™ Laminin Mouse Protein, Natural Cat. No. 08-774-271), 12-well (Fisher Scientific, Cat.
(Cat. No. 23017015) No. 08-774-270), or 6-well (Fisher Scientific, Cat. No.
08-774-268), or Gibco Poly-D-Lysine (to prepare poly-D-
• Optional: Gibco™ Poly-D-Lysine (Cat. No. A3890401) lysine–coated culture plates)

• Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent • 15 mL and 50 mL sterile polypropylene conical tubes
(Cat. No. A1110501): Thawed Accutase reagent can be
kept at 2°C to 8°C for up to 2 weeks. • 5, 10, 25, and 50 mL sterile pipettes

• Gibco™ Distilled Water (Cat. No. 15230162)

40
 Neural cell culture and differentiation

Preparing media Preparing matrix

To make 100 mL complete Neuronal Differentiation Medium To prepare the matrix, follow the process below to
with CultureOne Supplement, mix the components below apply coatings in the sequence of poly-D-lysine first and
under sterile conditions: laminin second.

Component Final conc. Amount Coating culture vessels with poly-D-lysine

Neurobasal Medium or 1X 96 mL 1. Dilute the poly-D-lysine solution in sterile DPBS,
Neurobasal Plus Medium[1] calcium, magnesium (DPBS +/+) to prepare a 50 μg/mL
B-27 Supplement (50X), 2% 2 mL working solution.
serum free or B-27 Plus
Supplement[2]
2. Coat the surface of the culture vessel with the
GlutaMAX Supplement 2 mM 1 mL
working solution of poly-D-lysine (e.g., 50 μL/well for a
CultureOne Supplement 1% 1 mL 96-well plate).
(100X)
Ascorbic acid (200 mM) 200 μM 100 μL
3. Incubate the vessel at room temperature for 1 hour.
1
Make complete medium with either Neurobasal Medium with B-27 Supplement or Neurobasal Plus
Medium with B-27 Plus Supplement. Do not interchange Neurobasal Plus Medium with Neurobasal
Medium and vice versa. 4. Remove the poly-D-lysine solution and rinse the
2
Supplement can be thawed at 2°C to 8°C overnight or quickly in a 37°C water bath for about culture surface 3 times with sterile distilled water (e.g.,
5 minutes, and then aliquoted and frozen at –20°C to –5°C to allow for the preparation of smaller
volumes of complete medium. Avoid repeated thawing and freezing. 100 μL/well for a 96-well plate).

Note: Complete medium can be stored at 2–8°C in the Make sure to rinse the culture vessel thoroughly, as
dark for up to 2 weeks. Warm medium in a 37°C water excess poly-D-lysine solution can be toxic to the cells.
bath for 5–10 minutes before using. Do not warm medium
in a 37°C water bath for >10 minutes, as this may cause 5. Remove the distilled water and leave the coated culture
degradation of the medium. vessel uncovered in the laminar hood to dry.

Optional: Add growth factors such as 10−20 ng/mL glial The culture surface should be fully dry after 2 hours.
cell–derived neurotrophic factor and 10−20 ng/mL brain-
derived neurotrophic factor to improve neuron survival. Note: Plates can be used immediately or stored at 4°C
once they are dry. For storage at 4°C, tightly wrap the
Optional: Add antibiotics such as gentamicin. vessel with Parafilm laboratory film and use within one
week of coating.

Coat culture plates with laminin

1. Thaw a vial of laminin stored at −80°C at room
temperature.

2. Dilute the thawed laminin solution 1:100 with sterile

distilled water.

41
Neural cell culture and differentiation

3. Add laminin solution into poly-D-lysine–coated 8. Gently shake the tube containing NSCs and add an
plates to cover the whole surface, and incubate in a appropriate amount of diluted NSC suspension into
37°C, 5% CO2 incubator for 1 hour. each well of culture plates to plate NSCs at a density of
5 x 104 cells/cm2 or less.
4. Culture plates can now be used. Just prior to use,
aspirate the laminin solution from each well. Cells can Note: If the passage number of NSCs is less than
be plated directly onto the laminin-coated plates without 5, add ROCK inhibitor Y-27632 into Neuronal
rinsing. Coated plates can also be stored at 2–8°C for Differentiation Medium to the final concentration of
up to one week. When storing, seal culture plates with 5 μM. The treatment with ROCK inhibitor at the time
Parafilm laboratory film to prevent drying. Before using, of NSC plating is crucial for cell survival with NSCs
warm up the coated plates stored at 2–8°C at room less than 5 passages. The optimal plating density may
temperature for 20–30 minutes. also vary depending on hPSC lines from which NSCs
were derived.
Plate and differentiate NSCs into neurons
9. Move the culture plates in several quick back-and-forth
1. Dissociate expanded hPSC-derived NSCs in culture with and side-to-side motions to disperse NSCs across the
StemPro Accutase Cell Dissociation Reagent or thaw surface, and place them gently in a 37°C CO2 incubator.
frozen hPSC-derived NSCs.
10. Add the same volume of pre-warmed Neuronal
2. Resuspend dissociated or thawed NSCs with 5−10 mL Differentiation Medium containing Neurobasal
DPBS, no calcium, no magnesium (DPBS –/–). Medium and B-27 Supplement into each well of plates
2−3 days after NSC plating, and return them into a
3. Centrifuge the cells at 300 x g for 5 minutes and aspirate 37°C CO2 incubator.
the supernatant.
Note: Differentiating neurons detach easily. When
4. Resuspend NSCs in 1−2 mL of pre-warmed Neuronal removing spent medium, do not touch cells with
Differentiation Medium containing Neurobasal Medium pipette tips. Also, add fresh medium gently toward the
and B-27 Supplement, depending on the number wall of culture plates.
of NSCs.
11. Change spent medium every 2−3 days thereafter. When
5. Determine the concentration of viable cells using your changing medium, remove half of spent medium from
preferred method. each well and add the same volume of pre-warmed
fresh Neuronal Differentiation Medium with Neurobasal
6. Dilute the NSC suspension with pre-warmed Neuronal Medium and B-27 Supplement into each well of plates
Differentiation Medium containing Neurobasal Medium and return them into a 37°C CO2 incubator.
and B-27 Supplement to an appropriate concentration.
12. Maintain neurons differentiated with CultureOne
7. Aspirate the laminin solution from poly-D-lysine– and Supplement for 1−5 weeks or longer, depending on
laminin-coated plates. NSC lines and the purpose of experiments.

42
 Neural cell culture and differentiation

Note: At 1−2 weeks after NSC differentiation, Expected results

CultureOne Supplement can be withdrawn by A B MAP2 SOX1 DAPI
adding fresh Neuronal Differentiation Medium without
CultureOne Supplement into each well of plates when
changing spent medium. However, withdrawal of
CultureOne Supplement may increase the chance of
cell clumps reforming in the culture due to proliferating
progenitor cells.

Note: Neuronal Differentiation Medium containing

C MAP2 SOX1 DAPI D Neurofilament Synaptophysin DAPI
Neurobasal Plus Medium and B-27 Plus Supplement
can be used to replace Neuronal Differentiation
Medium containing Neurobasal Medium and B-27
Supplement after 5 days of NSC plating for better
survival of differentiating neurons.

Figure 10-1. Differentiation of hPSC-derived NSCs by PSC Neural

Induction Medium into neurons. (A) H9 ESC-derived NSCs were
plated at a density of 5 x 104 cells/cm2 in Neuronal Differentiation Medium
containing B-27 Supplement and Neurobasal Medium. (B) Without
CultureOne Supplement, cells at 2 weeks of differentiation were highly
dense, formed cell clumps, and contained MAP2-positive neurons (green)
and a significant number of SOX1-positive NSCs (red). (C) At 2 weeks
of differentiation, cultures treated with CultureOne Supplement had an
even distribution of MAP2-positive neurons (green) with minimal SOX1-
positive NSCs (red) and no cell clumps. (D) At 5 weeks of differentiation,
differentiated cells treated with CultureOne Supplement expressed the
mature neuronal markers neurofilament (green) and synaptophysin (red).
The nuclei were counterstained with DAPI (blue) in panels B–D.

43
Neural cell culture and differentiation

11 PSC-derived neuron cell culture: Neuronal

differentiation, maturation, and maintenance

Summary Generating PSC-derived neurons

Human induced pluripotent stem cell (iPSC)-derived There are many ways to generate PSC-derived neurons.
neurons have increasingly become a valuable system for The most commonly used methods include the monolayer
the study of neurological disorders. Improved differentiation approach (Gibco™ PSC Neural Induction Medium), rosette
protocols, cell reprogramming, and gene editing enable formation, and factor-driven induction (Figure 11-1).
scientists to generate patient-specific, disease-in-a-dish
models for disorders such as Parkinson’s disease, PSC-derived neurons
Embryoid body Rosette
Alzheimer’s disease, and autism, among others. These
human models tend to be flexible and scalable, and
maintain many of the characteristics found in these
disorders, which are key requirements for their use in
mechanistic and drug discovery studies.
Pluripotent stem cell Neural stem cell

A critical step in generating useful PSC-derived neurons is

neuronal maturation. During maturation, neurons extend
neurites to form highly connected networks, express
Monolayer approach (PSC Neural Induction Medium)
synaptic markers, and generate spontaneous, networked
electrical activity. Robust maturation is necessary for PSC-
derived neurons to be relevant disease model systems. Factor-driven
Typical maturation conditions are inefficient, generating induction

poorly matured neurons with low levels of functionality.

Recently we developed a neuronal maturation and Neurons

maintenance medium, the Gibco™ B-27™ Plus Neuronal

Figure 11-1. Methods for generating PSC-derived neurons.
Culture System, which includes a user guide specifically Schematic of the three most common methods used for generating
for PSC-derived neurons. This next-generation system PSC-derived neurons.
was designed to improve long-term neuronal survival,
maturation, and functionality of neurons in culture. The monolayer and rosette differentiation methods mimic
in vivo development of PSC-derived neurons, and both
This protocol details neuronal differentiation, maturation, involve an intermediate step that yields an expandable
and maintenance of PSC-derived neurons. population of neural stem cells (NSCs) that can be further
differentiated into neurons. NSC methods are flexible and
View this protocol online and order products at may be more relevant to disease modeling.
thermofisher.com/b27plus
Another method is factor-driven induction of PSCs
into neuronal “iN” cells. This method typically involves
overexpression of lineage-specific factors to rapidly induce
a neuronal cell fate. The benefits of this system are rapid
and highly efficient generation of functional neurons within

44
 Neural cell culture and differentiation

14 days. Factor-driven induction can also be used to Required materials

generate induced neurons from somatic cells.
Media and reagents
• Gibco™ Poly-D-Lysine (Cat. No. A3890401)
Neuronal differentiation and maturation • Gibco™ Neurobasal™ Plus Medium (Cat. No. A3582901)

Starting from an NSC population, cells should be • Gibco™ B-27™ Plus Supplement (50X), serum free
differentiated toward a neuronal fate for 3–7 days. Optimal (Cat. No. A3582801)
conditions for neuronal differentiation of NSCs depend on
• Gibco™ GlutaMAX™ Supplement (Cat. No. 35050061)
the NSC derivation method. For example, NSCs from the
monolayer method require different media conditions than • Gibco™ DPBS, no calcium, no magnesium
NSCs from the rosette method. When differentiating cells (Cat. No. 14190144)
have adopted a neuronal-like morphology (Figure 11-2),
• Ascorbic Acid (MilliporeSigma, Cat. No. A8960)
cells are ready to switch to a B-27 Plus neuronal maturation
medium (B-27 Plus NMM) for continued maturation and • Gibco™ Distilled Water (Cat. No. 15230162)
long-term maintenance.
• Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent
This protocol provides guidance for differentiating (Cat. No. A1110501)
and maturing neurons from NSCs derived by the • Gibco™ Neurobasal™ Medium (Cat. No. 21103049)
monolayer method with PSC Neural Induction Medium.
Recommended medium conditions for neuronal • Gibco™ B-27™ Supplement (50X), serum free
differentiation of rosette-derived NSCs or the factor-driven (Cat. No. 17504044)
method can be found in the appendix of the user guide: • Gibco™ CultureOne™ Supplement (100X)
B-27 Plus Neuronal Culture System (PSC-Derived (Cat. No. A3320201)
Neuron Applications).

A Day 0 B Day 2 C Day 5 Preparing media

Ascorbic acid stock preparation

1. To prepare 200 mM ascorbic acid stock solution (500X),
add 20 mL of distilled water to 579.0 mg of ascorbic acid
powder and mix until dissolved.

NSC neuronal differentiation medium Ready for B-27 Plus 2. After dissolving, filter-sterilize through a 0.22 µm
neuronal maturation medium
filter, aliquot 100–200 µL into sterile tubes, and store
Figure 11-2. NSC differentiation: guidance for switching to the B-27 at –20°C.
Plus system for maturation.

45
Neural cell culture and differentiation

B-27 neuronal differentiation medium (B-27 NDM) Preparing matrix

Complete medium requires supplementation of Neurobasal
Medium with B-27 Supplement (50X), CultureOne To prepare the matrix, follow the process below to
Supplement, GlutaMAX Supplement, and ascorbic acid. apply coatings in the sequence of poly-D-lysine first and
Complete B-27 NDM is stable for 2 weeks when stored in laminin second.
the dark at 4°C.
Coating culture vessels with poly-D-lysine
To prepare 100 mL of complete medium, aseptically mix 1. Dilute the poly-D-lysine solution in sterile DPBS without
the following components. For larger volumes, increase the Ca2+ and Mg2+ (DPBS –/–) to prepare a 50 μg/mL
component amounts proportionally. working solution.

Component Final conc. Amount 2. Coat the surface of the culture vessel with the
Neurobasal Medium 1X 98 mL working solution of poly-D-lysine (e.g., 50 μL/well of a
B-27 Supplement (50X), 2% 2 mL 96-well plate).
serum free
CultureOne Supplement 1% 1 mL 3. Incubate the vessel at room temperature for 1 hour.
(100X)
GlutaMAX Supplement 1X 1 mL 4. Remove the poly-D-lysine solution and rinse the
Ascorbic acid (200 mM) 200 μM 100 μL culture surface 3 times with sterile distilled water (e.g.,
100 μL/well of a 96-well plate).
B-27 Plus neuronal maturation medium (B-27
Plus NMM) Make sure to rinse the culture vessel thoroughly as
Complete medium requires supplementation of Neurobasal excess poly-D-lysine solution can be toxic to the cells.
Plus Medium with B-27 Plus Supplement (50X), CultureOne
Supplement, GlutaMAX Supplement, and ascorbic acid. 5. Remove distilled water and leave the coated culture
Complete B-27 Plus NMM is stable for 2 weeks when vessel uncovered in the laminar hood to dry.
stored in the dark at 4°C.
The culture surface will be fully dry after 2 hours.
To prepare 100 mL of complete medium, aseptically mix
the following components. For larger volumes, increase the Note: Once the plates are dry, they can be used
component amounts proportionally. immediately or stored at 4°C. For storage at 4°C, tightly
wrap the vessel with Parafilm laboratory film and use
Component Final conc. Amount within one week of coating.
Neurobasal Plus Medium 1X 98 mL
B-27 Plus Supplement (50X), 2% 2 mL
serum free
CultureOne Supplement 1% 1 mL
(100X)
GlutaMAX Supplement 1X 1 mL
Ascorbic acid (200 mM) 200 μM 100 μL

46
 Neural cell culture and differentiation

Coating culture vessels with laminin 5. Determine the concentration of viable cells using your
6. Thaw a vial of laminin stock solution at 4°C. preferred method.

Note: Upon receipt, laminin stock solution should be 6. Dilute the NSC suspension with pre-warmed B-27 NDM
aliquoted and stored at –80°C to avoid repeated to an appropriate concentration.
thawing and freezing.
7. Remove precoated poly-D-lysine– and laminin-coated
7. To create a working solution, dilute the laminin stock plates from the incubator. Just prior to use, aspirate
solution to a concentration of 3 μg/mL in sterile the laminin solution from each well and rinse once with
distilled water. DPBS –/–, before plating cells.

8. Add the laminin solution to poly-D-lysine–coated plates 8. Gently mix the tube containing the NSCs and add an
to cover entire surface. For example, to coat a 96-well appropriate amount of the diluted NSCs in B-27 NDM
plate, add 60 μL of the dilute 3 μg/mL laminin solution. suspension into each well of culture plate.
Incubate plates in a humidified 37°C, 5% CO2 incubator
for 2 hours, or tightly wrap the plate with Parafilm 9. Move the culture plates in several quick back-and-forth
laboratory film and store overnight at 4°C. and side-to-side motions to disperse NSCs across the
surface, and place them gently in a humidified 37°C,
9. Immediately prior to use, aspirate the laminin solution 5% CO2 incubator for 48 hours.
from each well, rinse once with DPBS –/–, and then
plate cells. 10. After the 48-hour incubation (on day 2), perform a half-
medium change with B-27 NDM.

Differentiating and maturing neurons 11. At day 5, differentiating cells will have adopted a
from NSCs derived using PSC Neural neuronal-like morphology (see Figure 11-2 on page 45),
and are ready for neuronal maturation.
Induction Medium
Plating and differentiating NSCs Maturation and maintenance of PSC-derived neurons
1. Dissociate expanded hPSC-derived NSCs in culture with 12. Once cells adopt a neuronal morphology (see Figure
StemPro Accutase Cell Dissociation Reagent, or thaw 11-2 on page 45), remove half the spent medium and
frozen hPSC-derived NSCs. replace with an equal volume of pre-warmed, fresh
complete B-27 Plus NMM.
2. Resuspend dissociated or thawed NSCs with
5−10 mL DPBS –/–. 13. Change spent medium every 3–4 days thereafter. (For
high-density cultures, change medium every 2–3 days).
3. Centrifuge the cells at 300 x g for 5 minutes and aspirate When changing medium, remove half the spent medium
the supernatant. from each well and add the same volume of pre-warmed
fresh B-27 Plus NMM into each well of plates and return
4. Resuspend NSCs in 1−2 mL of pre-warmed B-27 NDM, cultures to a humidified 37°C, 5% CO2 incubator.
depending on the number of NSCs.

47
Neural cell culture and differentiation

Neuron survival
200

Avg. HuC/D-positive cells per field

160

120

80

40
14. Change spent medium every 3–4 days thereafter. (For Expected results
high-density cultures, change medium every 2–3 days). 0
22 35 49
Increased neuronal survival with the
Time (days)
15. Maintain maturing neurons with B-27 Plus NMM for B-27 Plus system B-27 classic system B-27 Plus system
3–10 weeks or longer, depending on NSC lines and the
purpose of experiments. B-27 system B-27 Plus system

Note: Differentiated neurons detach easily. When

removing spent medium, do not touch cells with pipet
tip; when adding fresh medium, do it gently toward the
wall of the culture plate.

 HuC/D  MAP2  DAPI  HuC/D  MAP2  DAPI

Neuron survival
200
Avg. HuC/D-positive cells per field

160

120

80

40

0
22 35 49
Time (days)

B-27 classic system B-27 Plus system

B-2711-3.
Figure system B-27 Plus
The B-27 Plus system increases system
long-term survival
of iPSC-derived neurons. NSCs derived using PSC Neural Induction
Medium matured in the B-27 Plus system for 4 weeks, resulting in an
approximately 2-fold increase in survival compared to classic B-27
Supplement and Neurobasal Medium.

 HuC/D  MAP2  DAPI  HuC/D  MAP2  DAPI

48
 Synapsin expression Neural cell culture and differentiation
1,400
Synapsin-positive area per neuron

1,200

1,000

800

600

400

200

0
22 35 49
Enhanced neuronal maturation with the B-27
Time (days)
Improved physiological activity with the B-27
Plus system B-27 classic system B-27 Plus system
Plus system

B-27 system B-27 Plus system A B-27 Plus system

 Synapsin 1/2  MAP2  DAPI  Synapsin 1/2  MAP2  DAPI 0 100 200 300 400 500 600

Time (sec)
Synapsin expression
1,400 B B-27 classic system
Synapsin-positive area per neuron

1,200

1,000

800

600

400

200 0 100 200 300 400 500 600

0 Time (sec)
22 35 49
Time (days)
Figure
C 11-5. The B-27BrainPhys
Plus system SM1
improves functional activity in
system
B-27 classic system B-27 Plus system neurons from monolayer NSCs derived using PSC Neural Induction
Medium. Multi-electrode array (MEA) data recorded as raster plots from
B-27
Figure system
11-4. B-27 Plus
The B-27 Plus system enhances system
synaptic marker NSCs matured in the B-27 Plus system for 31 days (A) showed high levels
expression in iPSC-derived neurons. NSCs that were derived using PSC of spontaneous, synchronous network bursting activity compared to the
Neural Induction Medium and then matured in the B-27 Plus system for 7 classic B-27 culture system (B). This networked activity was maintained for
weeks resulted in significantly higher levels of synapsin 1/2 expression than over 4 weeks.
in the classic B-27 system. Synaptic marker expression is an indicator of
functional maturity.

0 100 200 300 400 500 600

Time (sec)
 Synapsin 1/2  MAP2  DAPI  Synapsin 1/2  MAP2  DAPI

49
Neural cell culture and differentiation

12 Differentiating PSCs to midbrain dopaminergic

neurons

• Gibco™ DMEM/F-12, GlutaMAX™ Supplement

Summary (Cat. No. 10565018) (base medium for maturation)

Midbrain dopaminergic (mDA) neurons derived from • Gibco™ Laminin Mouse Protein, Natural
human pluripotent stem cells (hPSCs) provide an excellent (Cat. No. 23017015)
alternative to primary human neurons for disease modeling • Gibco™ DPBS, no calcium, no magnesium
of Parkinson’s disease and drug screening. During brain (Cat. No. 14190144)
development, mDA neurons are derived from distinct
• Gibco™ Distilled Water (Cat. No. 15230162)
populations of cells termed midbrain floor plate (mFP) cells.
In this protocol, we describe how to (1) specify hPSC to • Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent
mFP cells, (2) expand and cryopreserve specified cells, and (Cat. No. A1110501)
(3) revive and mature cells to mDA neurons.
• ROCK inhibitor Y-27632 (MilliporeSigma, Cat. No. Y0503)

View this protocol online and order products at • Dimethyl sulfoxide, Hybri-Max™ grade (DMSO)
thermofisher.com/dopadiff (MilliporeSigma, Cat. No. D2650)

• Gibco™ Human Dopaminergic Neuron

Immunocytochemistry Kit (Cat. No. A29515) (for image-
Required materials based analysis of intermediate floor plate progenitors and
mature dopaminergic neurons)
Cells
• Gibco™ Human Episomal iPSC Line (Cat. No. A18945) Equipment and plasticware
or equivalent PSCs cultured in Gibco™ Essential 8™ or • Thermo Scientific™ Nunclon™ Sphera™ Flasks
Essential 8™ Flex Medium (see Cat. No. information below) (Cat. No. 174951 or 174952) (for suspension culture)

• Thermo Scientific™ Nunclon™ Sphera™ Dishes

Media and reagents (Cat. No. 174932) (for suspension culture)
• Gibco™ PSC Dopaminergic Neuron Differentiation Kit
(Cat. No. A3147701: Contains Floor Plate Specification • Gibco™ Poly-D-Lysine (Cat. No. A3890401) (to prepare
Supplement (20X) stored at –20°C to –5°C in the dark; poly-D-lysine and laminin double-coated culture plates)
Floor Plate Cell Expansion Kit, consisting of Floor Plate
• Thermo Scientific™ Mr. Frosty™ Freezing Container
Cell Expansion Base stored at 4°C and Floor Plate (Cat. No. 5100-0001)
Cell Expansion Supplement (50X) stored at –20°C to
–5°C in the dark; and Dopaminergic Neuron Maturation • Thermo Scientific™ Nalgene™ General Long-Term Storage
Supplement (50X) stored at –20°C to –5°C in the dark.) Cryogenic Tubes (Cat. No. 5000-0012 or 5000-1012) (for
banking floor plate progenitor cells)
• Gibco™ Essential 8™ Medium (Cat. No. A1517001),
contains Basal Medium and Supplement; or Essential 8™ • 37°C humidified cell culture incubator with 5% CO2
Flex Medium Kit (Cat. No. A2858501), contains Flex Basal
Medium and Flex Supplement • Liquid nitrogen storage

• Gibco™ Vitronectin (VTN-N), Recombinant Human • Centrifuge

Protein, Truncated (Cat. No. A14700) • 37°C water bath
• Gibco Neurobasal Medium (Cat. No. 21103049) (base
™ ™
• 15 and 50 mL sterile polypropylene conical tubes
medium for specification)
• 5, 10, 25, and 50 mL sterile pipettes

50
 Neural cell culture and differentiation

Preparing media

Specification medium Maturation medium

Floor Plate (FP) Specification Supplement (20X) can be Dopaminergic (DA) Neuron Maturation Supplement (20X)
thawed at 4°C or room temperature and dispensed into can be thawed at 4°C or room temperature and dispensed
aliquots if desired. Complete medium is stable for 2 weeks into aliquots if desired. Complete medium is stable for
when stored in the dark at 2°C to 8°C. 2 weeks when stored in the dark at 2°C to 8°C.

To prepare 100 mL of FP specification medium, mix the To prepare 100 mL of DA neuron maturation medium,
following components under sterile conditions. For larger mix the following components under sterile conditions.
volumes, increase the component amounts proportionally. For larger volumes, increase the component amounts
proportionally.
Final
Component Amount
conc. Component Final Amount
Neurobasal Medium 1X 95 mL conc.
FP Specification Supplement (20X) 1X 5 mL DMEM/F-12 1X 98 mL
DA Neuron Maturation Supplement 1X 2 mL
(50X)
Expansion medium
Floor Plate (FP) Cell Expansion Supplement (20X) can be
thawed at 4°C or room temperature and dispensed into ROCK inhibitor stock solution
aliquots if desired. Complete medium is stable for 2 weeks To prepare 10 mM ROCK inhibitor Y-27632 solution,
when stored in the dark at 2°C to 8°C. add 10 mg of Y-27632 to 3.125 mL of distilled water. Mix
well until dissolved.
To prepare 100 mL of FP expansion medium, mix the
following components under sterile conditions. For larger After dissolving, filter-sterilize through a 0.22 µm filter,
volumes, increase the component amounts proportionally. aliquot 20–50 µL into sterile tubes, and store at –20°C in
the dark for up to 1 year. Thawed Y-27632 solution can be
Component Final Amount kept at 4°C for up to 4 weeks.
conc.
FP Cell Expansion Base 1X 98 mL
FP Cell Expansion Supplement (50X) 1X 2 mL

51
Neural cell culture and differentiation

Preparing matrix

Vitronectin-coated plates Note: Laminin may form a gel when thawed too rapidly.
1. Prepare a 1:50 dilution of vitronectin solution in DPBS, Thawing the stock solution in the cold (4°C) prevents
no calcium, no magnesium (DPBS –/–) for a final this. Thawed stock solution can be stored at 4°C for
concentration of 10 μg/mL. up to 1 month.

2. Add 1 mL of the diluted vitronectin solution to each well Poly-D-lysine and laminin double-coated plates
of a 6-well plate. 1. Coat each well of a 6-well plate with 1 mL of
poly-D-lysine working solution (100 μg/mL).
3. Incubate the coated plates at room temperature for
1 hour. The culture vessel can now be used or stored 2. Incubate the coated plates at room temperature for
at 4°C wrapped in Parafilm laboratory film for up to 1–2 hours.
one week. Do not allow the vessel to dry.
3. Remove the Poly-D-Lysine solution and rinse 3 times
4. Before use, pre-warm the culture vessel to room with distilled water.
temperature for at least 1 hour before aspirating and
discarding the vitronectin solution. 4. Prepare a 15 μg/mL working solution of laminin in
sterile distilled water.
Note: It is not necessary to rinse off the culture plate
after the removal of the vitronectin solution. 5. Remove distilled water rinse and leave the coated culture
vessel uncovered in the laminar hood to dry.
Laminin-coated plates
1. Thaw the required volume of 1.0 mg/mL laminin stock 6. Add 1 mL of the 15 μg/mL laminin working solution to
solution (stored at –80°C) slowly at 4°C. each well of a 6-well plate.

2. Prepare a 1:100 dilution of laminin solution in water for a 7. Incubate the coated plates overnight at 4°C or at 37°C
final concentration of 10 μg/mL. for 2 hours.

3. Add 1 mL of the diluted laminin solution to each well 8. Before use, pre-warm the culture vessel to room
of a 6-well plate. temperature for at least 1 hour before aspirating and
discarding the laminin solution.
4. Incubate the coated plates at 4°C overnight or at 37°C
for 2 hours. The culture vessel can now be used or Note: You can use the coated culture plate immediately
stored at 4°C wrapped in Parafilm laboratory film for up or store it at 4°C wrapped in Parafilm laboratory film for
to one week. Do not allow the vessel to dry. up to one week. Do not allow the plate to dry.

5. Before use, pre-warm the culture vessel to room

temperature for at least 1 hour before aspirating and
discarding the laminin solution.

52
 Neural cell culture and differentiation

Specification (PSC to mFP) Expansion

Set up hPSC culture (day –1) Harvest FP progenitor cells (day 10, 12, and 16)
1. Prepare vitronectin-coated plate and complete 1. Prepare laminin-coated plate and FP cell
Essential 8 Medium. expansion medium.

2. Plate a high-quality PSC culture from frozen vial or 2. Aspirate the spent medium from the specification
ongoing culture on vitronectin plate in Essential 8 culture plate and rinse the wells with DPBS,
Medium to target 20–40% confluency on the next day. calcium, magnesium (DPBS +/+) to remove any
remaining medium.
3. If plated as single cells, supplement medium with 10 μM
ROCK inhibitor Y-27632 to inhibit cell death. 3. Aspirate the DPBS and add an appropriate volume of
StemPro Accutase Cell Dissociation Reagent to fully
Note: Depending on cell line used, culture kinetics cover the surface (1 mL per well of a 6-well plate, or
are different. Optimization is needed to find the right 1 mL per 10 cm2 of surface area).
seeding density to get 100% confluency after 6–7 days
of specification. With H9 ESC lines, 30,000 cells/cm2 4. Incubate the vessel at 37°C, 5% CO2 for ~5–15 minutes,
seeding density resulted in 20–40% confluency on continually observing the wells for cell detachment.
the next day.
5. After several minutes or when some colonies start
Specification (day 0–day 10) detaching (whichever happens first), gently tap the
1. Start specification by changing medium with FP bottom of the vessel several times. Most colonies
specification medium (day 0). Aspirate the spent should freely come into suspension. If all colonies do not
Essential 8 Medium containing the ROCK inhibitor and detach, wait 1–2 minutes, and then tap the vessel again
replace it with pre-warmed FP specification medium. to detach the remaining colonies.

2. Incubate at 37°C in a humidified atmosphere of 6. Transfer the cell clumps to a sterile 50 mL culture tube.
5% CO2 in air.
7. Rinse the wells of the specification culture plate twice
3. Replenish culture with fresh medium at day 3, 5, 7, with DPBS +/+, using 4x the volume of StemPro
and 9. Medium consumption will be increased over Accutase reagent used in each well (4 mL per well of
time, so use 2X volume for later points (day 7 and 9) a 6-well plate). After each rinse, collect the cell clumps
to compensate. in the same 50 mL culture tube to ensure the recovery
of all colonies.

8. Centrifuge the cell suspension at 300 x g for 3 minutes

at 4°C to pellet the cells. Carefully aspirate the
supernatant, leaving the cell pellet in the culture tube.

53
Neural cell culture and differentiation

Replate FP cells (day 10 and 12) with isopropyl alcohol, and freeze them at
1. Gently flick the bottom of the tube to dislodge the –80°C overnight.
cell pellet, and resuspend the cells in a sufficient
volume of complete expansion medium plus 5 μM 6. The next day, transfer the frozen vials to liquid nitrogen
ROCK inhibitor Y-27632. (vapor phase) for long-term storage.

2. Use 1:2 split ratio (i.e., one plate to two plates) for FP Recover frozen FPp2 cells (day 16)
passage 0 (FPp0) (day 10) and use 1:4 split ratio for FP 1. Remove the cryogenic vial of FPp2 cells from the
passage 1 (FPp1) (day 16). liquid nitrogen storage and immediately immerse it in
a 37°C water bath without submerging the cap. Swirl
Note: Overnight treatment with the ROCK inhibitor the vial gently.
is required upon passaging. The ROCK inhibitor is
removed from the culture the following day when the 2. When only an ice crystal remains (~1–2 minutes), remove
spent medium is replaced with FP specification medium. the vial from the water bath and spray the outside of it
with 70% ethanol to decontaminate.
3. Incubate the cells overnight at 37°C in a humidified
atmosphere of 5% CO2. 3. Pipet the cells gently into a sterile 15 mL conical tube
using a 1 mL pipette.
4. The next day, replenish culture with fresh medium and
every other day thereafter. 4. Add 1 mL of DPBS –/– into the vial to collect the
remaining cells and transfer the cell suspension
Cryopreserve FP passage 2 (FPp2) cells (day 16) dropwise to the 15 mL conical tube. While adding,
1. Prepare freezing medium at 2X concentration gently move the tube back and forth to mix the cells and
(80% FP expansion medium + 20% DMSO) and chill prevent osmotic shock.
at 4°C before use.
5. Add an additional 3 mL of DPBS –/– to the cells to have
2. Calculate the volume of cells in the FPp2 cell suspension a 5 mL suspension.
that corresponds to the number of cells you want to
cryopreserve, and transfer to a sterile tube. 6. Remove a small volume of cell suspension and perform
a viable cell count.
3. Dilute the cells to 2X the intended final frozen
concentration using FP expansion medium at 4°C. 7. Centrifuge the cell suspension at 300 x g for 3 minutes
to pellet the cells. Carefully aspirate the supernatant,
4. In a dropwise manner, add the same volume of 2X leaving the cell pellet in the culture tube.
freezing medium (chilled to 4ºC) as the cell suspension
while gently rocking the tube back and forth. 8. Gently flick the bottom of the tube to dislodge
the cell pellet and resuspend the cells to
5. Aliquot 1 mL of the cell suspension into each cryogenic 1.0 x 106 viable cells/mL in FP expansion medium plus
vial, place the vials in a Mr. Frosty Freezing Container 5 μM ROCK inhibitor Y-27632.

54
 Neural cell culture and differentiation

Sphere formation of FPp2 (day 16–day 21) Maturation

1. On day 16, harvest or thaw FPp2 cells as described,
then remove a small volume of cells and perform a Dissociate spheres (day 21)
viable cell count. 1. Prepare double-coated culture plates and DA
maturation medium.
2. Resuspend the FPp2 cells to 1.0 x 106 viable cells/mL
in FP expansion medium plus 5 μM ROCK 2. Transfer the sphere suspension from culture vessel to a
inhibitor Y-27632. sterile 15 mL conical tube. Allow spheres to settle to the
bottom of the tube (~2–5 minutes) before proceeding to
3. Transfer cell suspension to a non–tissue culture treated the next step.
vessel and adjust the volume of the cell suspension to
the size of vessel. 3. Carefully aspirate the spent medium, leaving the spheres
at the bottom of tube in a minimal volume (~100 μL) of
4. Incubate the cells overnight at 37°C in a humidified the remaining medium. Resuspend the spheres in 5 mL
atmosphere of 5% CO2. of DPBS –/–.

5. Perform a complete medium change by the 4. Repeat steps 2 and 3, leaving the spheres at the bottom
centrifugation method on the next day and every other of tube in a minimal volume (~100 μL) of DPBS.
day thereafter. Transfer the spheres to a 15 mL conical
tube and then centrifuge at 200 x g for 2 minutes. 5. Add 1 mL of StemPro Accutase Cell Dissociation
Aspirate the supernatant and discard. Reagent to the spheres and incubate for 30 minutes at
37°C. Every 10 minutes, gently swirl the cell suspension
6. Resuspend the spheres in fresh FP expansion to ensure that spheres are exposed to the StemPro
medium without the ROCK inhibitor, and then transfer Accutase reagent evenly.
to original flask.
6. While the spheres are incubating with the dissociation
7. Pipet the sphere suspension up and down several reagent, aliquot the amount of complete DA maturation
times to prevent them from merging with each medium needed for the day and warm at 37°C.
other before plating.
7. Gently pipet the cell suspension up and down with a
P1000 pipette until all of the spheres are dispersed into
a single-cell suspension.

8. Remove a small volume of cell suspension to perform

a viable cell count using an automated cell counter
(e.g., Invitrogen™ Countess™ II Automated Cell Counter)
or a hemocytometer.

55
Neural cell culture and differentiation

9. Centrifuge the cell suspension at 300 x g for 3 minutes 3. Incubate the cells overnight at 37°C in a humidified
to pellet the cells. Carefully aspirate the supernatant, atmosphere of 5% CO2.
leaving the cell pellet in the culture tube.
4. On day 22 of differentiation (first medium change),
Plate and mature FP cells to DA neurons add the same volume of fresh DA maturation medium
(day 21–day 35) (without the ROCK inhibitor) as the existing culture
1. Resuspend the cell pellet to a single-cell suspension volume (e.g., 2 mL for each well of a 6-well plate).
in DA maturation medium plus 5 μM ROCK
inhibitor Y-27632. 5. For subsequent feeds (every 2~3 days), aspirate
half of the spent medium and replace it with fresh
2. Seed the double-coated culture plates with the DA maturation medium.
dissociated cells at a seeding density of 1.0 x 105
to 2.0 x 105 cells/cm2 in DA maturation medium plus
5 μM ROCK inhibitor Y-27632.

A FOXA2 B OTX2 C FOXA2 OTX2 DAPI

D LMX1A E SOX1 F LMX1A SOX1 DAPI

Figure 12-1. Marker expression of induced FP progenitor cells. hPSCs were treated with FP
specification medium for 7 days, and the cells were analyzed for the key phenotypic markers of the
human DA neuron lineage using the Human Dopaminergic Neuron Immunocytochemistry Kit (Cat.
No. A29515). (A–C) After FP specification of hPSCs, the cells express FP marker FOXA2 (green) and
rostral marker OTX2 (red). (D–E) The specified FP cells are positive for the DA progenitor marker
LMX1A (green), but negative for the neural stem cell marker SOX1 (red).

56
 Neural cell culture and differentiation

A TH B FOXA2 DAPI C TH FOXA2

Figure 12-2. Representative images of mature DA neurons after 14 days in DA maturation

medium. The majority of the TH-expressing neurons also coexpressed FOXA2. (A) Anti-TH antibody
(green). (B) Anti-FOXA2 antibody (red) and Invitrogen™ NucBlue™ reagent (a DAPI nuclear DNA stain)
(blue). (C) Merged image with anti-TH and anti-FOXA2 antibodies (green and red).

57
Neural cell culture and differentiation

13 Culture of cortical astrocytes

Summary

Astrocytes constitute a critical mass of the central nervous Sources of primary cortical astrocytes
system (CNS), in addition to oligodendrocytes and neurons. Gibco™ Rat Primary Cortical Astrocytes are isolated from
They are involved in adult CNS homeostasis, biochemical the cortices of fetal Sprague-Dawley rats at embryonic
and nutritional support of neurons and endothelial cells day 19 (E19) of gestation. The cells are isolated from tissue
that form the blood–brain barrier, perform the vast under sterile conditions, and placed through one round of
majority of synaptic glutamate uptake, and maintain enzymatic dissociation and expansion in astrocyte growth
extracellular potassium levels. Astroglial dysfunction has medium (85% DMEM containing 4.5 g/L glucose and
been implicated in a number of CNS pathologies. This 15% FBS). The cells are cryopreserved at passage 1 (P1) in
protocol describes the preparation of primary cortical 90% astrocyte growth medium plus 10% DMSO. Each vial
astrocytes from newborn rats or mice, or from human fetal of Rat Primary Cortical Astrocytes contains 1 x 106 cells/mL
brain tissue. that can be expanded in culture for at least one passage.

View this protocol online and order products at Gibco™ Human Astrocytes are derived from human
thermofisher.com/neuroprotocol/astro brain glial progenitors. They have star-like morphology
and express glial fibrillary acidic protein (GFAP) at
Introduction high percentages. Human Astrocytes are supplied
Astrocytes outnumber neurons by up to tenfold, and cryopreserved at a concentration of ≥1 x 106 cells/mL
have critical roles in adult CNS homeostasis (Pekny and in Gibco™ Astrocyte Medium without EGF and with
Nilsson, 2005). They provide biochemical and nutritional 10% DMSO.
support of neurons and endothelial cells that in turn
form the blood–brain barrier, perform synaptic glutamate Characteristics of Gibco Rat Primary
uptake, and maintain extracellular potassium (Rothstein Cortical Astrocytes
et al., 1996; Rothstein et al., 1994). Astroglial dysfunction • Isolated from the brain cortex of fetal Sprague-Dawley
has been implicated in a number of CNS pathologies rats at E19 of gestation
including amyotrophic lateral sclerosis (ALS) and ischemic
• Exhibit ≥70% viability upon thawing
neuronal death (Maragakis and Rothstein, 2006; Takano
et al., 2009). Transplant-based astrocyte replacement • Stain >80% positive for the astrocyte-specific
therapy has been shown to be a promising therapeutic marker GFAP
strategy against neuronal death (Lepore et al., 2008) and
in lessening the disease impact in ALS. Although there are • Stain ≤10% positive for neuron- and oligodendrocyte-
few known differences between cortical and hippocampal specific markers galactocerebroside (GalC) and
astrocytes, it has been reported that astrocytes from doublecortin (DCX)
different regions of the brain show different sensitivity to
• Exhibit a doubling time of approximately 9 days at P2
ischemic injury (Xu et al., 2001; Zhao and Flavin, 2000).
• Expandable in culture for at least one passage

58
 Neural cell culture and differentiation

• Gibco™ Trypan Blue Solution, 0.4% (Cat. No. 15250061)

Characteristics of Gibco Human Primary
(included with the Invitrogen™ Countess™ II Automated Cell
Cortical Astrocytes
Counter) or Invitrogen™ LIVE/DEAD™ Cell Vitality Assay
• Derived from progenitor cells isolated from human
Kit, C12 Resazurin/SYTOX™ Green (Cat. No. L34951)
fetal brain
• Gibco™ B-27™ Plus Neuronal Culture System
• Limited expansion capacity in culture
(Cat. No. A3653401: This kit contains B-27 Plus
Supplement (50X), stored at –20 to –5°C in the dark; and
Neurobasal Plus Medium, stored at 2–8°C in the dark.)
Required materials
• Gibco™ poly-D-lysine (Cat. No. A3890401)
Cells
• Gibco™ GlutaMAX™ Supplement (Cat. No. 35050061)
• Gibco™ Human Astrocytes (Cat. No. N7805100)
or Gibco™ Rat Primary Cortical Astrocytes
(Cat. No. N7745100) Equipment and plasticware
• Invitrogen™ Countess™ II Automated Cell Counter
Media and reagents (Cat. No. AMQAX1000) or hemocytometer
• Gibco™ Astrocyte Medium (Cat. No. A1261301: This
• 37°C incubator with humidified atmosphere of 5% CO2
kit contains N-2 Supplement, 100X, stored at –20°C;
Dulbecco’s Modified Eagle Medium (D-MEM) (1X), stored • Uncoated, tissue-culture treated flasks, plates, or
at 2°C to 8°C; and Gibco™ One Shot™ Fetal Bovine petri dishes
Serum, Certified, stored at –20°C in the dark.)
• Disposable, sterile 15 mL or 50 mL conical tubes, pre-
• Gibco™ Penicillin-Streptomycin (5,000 U/mL) rinsed with medium
(Cat. No. 15070063)

• Gibco™ EGF Recombinant Human Protein

(Cat. No. PHG0314) Preparing reagents and media
• Gibco™ DPBS, no calcium, no magnesium Complete astrocyte medium
(Cat. No. 14190144) Astrocyte Medium has been specifically formulated for
the growth and maintenance of human and rat astrocytes
• Gibco™ Geltrex™ LDEV-Free Reduced Growth Factor
while retaining their phenotype. The medium has three
Basement Membrane Matrix (Cat. No. A1413201)
components: basal medium (DMEM), N-2 Supplement, and
• Gibco™ StemPro™ Accutase™ Cell Dissociation Reagent One Shot Fetal Bovine Serum (FBS). Epidermal growth factor
(Cat. No. A1110501), pre-warmed to 37°C (EGF) may also be added to enhance astrocyte proliferation.

To prepare 100 mL of complete astrocyte medium, mix the

following components under aseptic conditions. For larger
volumes, increase the component amounts proportionally.

59
Neural cell culture and differentiation

Component Amount 1. Thaw a bottle of Geltrex Basement Membrane Matrix at

DMEM 88 mL 2°C to 8°C overnight.
N-2 Supplement 1 mL
FBS 10 mL 2. On ice, prepare a stock solution of Geltrex matrix diluted
Penicillin-Streptomycin 1 mL 1:1 in DMEM. Store in aliquots at –20°C until needed.
Optional: EGF (prepared as 100 μg/mL stock) 20 μL
3. Dilute the stock solution 1:100 in DMEM and coat the
Complete neuronal medium bottom of each culture vessel (200 μL of Geltrex matrix
The B-27 Plus Neuronal Culture System is a next-generation per cm2 of culture vessel).
media system that provides the highest rate of in vitro survival
of primary rodent and human stem cell–derived neurons. 4. Incubate the culture vessel at 36°C to 38°C for 1 hour.
Composed of B-27 Plus Supplement (50X) and Neurobasal
Plus Medium, this system is an evolution of the most cited Dishes coated with Geltrex matrix can be used immediately
neuronal cell culture products, Gibco™ B-27 Supplement or stored at 2°C–8°C for up to one week, sealed with
and Gibco™ Neurobasal Medium. It features an optimized Parafilm laboratory film. Do not allow dishes to dry out.
formulation, upgraded manufacturing process, and more
stringent quality control of raw materials and final product. Note: Warm stored Geltrex matrix plates to room
These improvements enable the highest neuronal cell temperature for 1 hour prior to adding astrocytes.
survival, improved electrophysiological activity, and enhanced
functional maturity compared to other neuronal cell culture Note: When you are ready to add cells, aspirate the Geltrex
media systems. matrix solution and rinse once with DPBS with calcium and
magnesium before adding the cell solution.
To prepare 100 mL of complete neuronal medium, mix the
following components under aseptic conditions. For larger
volumes, increase the component amounts proportionally. Thawing cryopreserved rat or human
primary cortical astrocytes
Component Amount
Neurobasal Plus Medium 98 mL Before thawing or harvesting Gibco Human Astrocytes,
B-27 Plus Supplement 2 mL prepare culture vessels coated with Geltrex matrix as
GlutaMAX Supplement 0.25 mL described previously.

Prepare plasticware coated with Geltrex Important: Astrocytes readily stick to plastics. Prewet all
matrix (human astrocytes only) plastics with complete astrocyte medium.

Before thawing or passaging Gibco Human Astrocytes, Note: We recommend seeding cells at 2 x 104 cells/cm2 for
prepare culture vessels coated with Geltrex matrix as rat astrocytes, or 4 x 104 cell/cm2 for human astrocytes.
described below. This is equivalent to 180,000 or 360,000 cells in 2–3 mL in
one well of a 6-well plate.
Note: Rat astrocytes do not require the use of Geltrex matrix–
coated plates.

60
 Neural cell culture and differentiation

1. Remove a vial of cells from liquid nitrogen storage 10. Adjust the cell density with warm complete astrocyte
and immediately thaw by swirling in a 37°C water medium for correct plating density.
bath. Remove the vial when the last bit of ice has
melted, typically <3 minutes. Do not submerge the vial 11. For human astrocytes, remove a Geltrex matrix–coated
completely, thaw for longer than 3 minutes, or create plate from 2°C to 8°C storage and warm to room
bubbles in the cell suspension, as this will decrease temperature for 1 hour. Remove the medium by tipping
cell viability. slightly to aspirate the Geltrex matrix solution.

2. When thawed, disinfect the outside of the tube with Note: Do not allow the plate surface to dry out before
70% isopropyl alcohol and transfer the tube to a laminar plating the cells. (Rat astrocytes do not require Geltrex
flow hood. matrix–coated plates.)

3. Precondition (prewet) a 15 mL centrifuge tube with warm 12. Immediately plate the cells at 2 x 104 cells/cm2 for rat
complete astrocyte medium. Discard the medium. astrocytes, or 4 x 104 cells/cm2 for human astrocytes.
This is equivalent to 180,000 or 360,000 cells in 2–3 mL
4. Using a prewetted sterile pipette tip, slowly transfer in one well of a 6-well plate.
the thawed cells (~1 mL) to the preconditioned
centrifuge tube. 13. Incubate the cells at 36°C–38°C in a humidified
atmosphere (90%) of 4–6% CO2 in air. Allow the cells to
5. Add 1 mL of medium to rinse the cryovial. Add this adhere for at least 24 hours.
dropwise to the centrifuge tube while swirling.
Note: Change the medium every 2 days.
6. Add 3 mL of additional warm complete astrocyte
medium slowly for a total of 5 mL.
Expanding rat primary cortical astrocytes
7. To remove cryoprotectant (DMSO) from the cells,
centrifuge the tube at 250 x g for 5 minutes. Remove 1. Remove the spent growth medium from the culture dish
and discard the supernatant above the cell pellet. containing the cells, and store in a sterile tube to use as
a washing solution.
8. Prewet a sterile pipette and suspend the cells in 2–3 mL
of warm complete astrocyte medium. 2. Rinse the surface of the cell layer once with DPBS
without Ca2+ and Mg2+ (approximately 2 mL DPBS per
9. Determine the viable cell count using your method of 10 cm2 culture surface area) by adding the DPBS to the
choice (e.g., Countess II Automated Cell Counter) to side of the vessel opposite the attached cell layer, and
seed at the correct density. rocking back and forth several times.

Note: If recovery seems poor, count the cells before 3. Aspirate the DPBS and discard.
and after centrifugation with the next vial to determine
if cells are lost due to centrifugation.

61
Neural cell culture and differentiation

4. To detach the cells, add 3 mL of pre-warmed Expanding human primary

StemPro Accutase Cell Dissociation Reagent per T-75 cortical astrocytes
flask; adjust volume accordingly for culture dishes
of other sizes. For replating human astrocytes, prepare culture vessels
coated with Geltrex matrix as described above.
5. Incubate for up to 30 minutes at 37°C. Rock the cells
every 5 minutes, and check for cell detachment and Equilibrate stored plates to room temperature for 1 hour prior
dissociation toward single cells under the microscope. to use.

6. Once you observe cell detachment, gently pipette up 1. Warm complete astrocyte medium and StemPro
and down to break clumps into a single-cell suspension. Accutase Cell Dissociation Reagent in a 37°C water bath
Stop the cell dissociation reaction by an adding equal before use.
volume of the spent medium from step 1. Disperse
the medium by pipetting over the cell layer surface 2. Transfer conditioned medium from the cells to a new
several times. tube; this will be used to stop the enzyme reaction in
step 6.
7. Transfer the cells to a new 15 mL or 50 mL pre-
rinsed conical tube, and centrifuge at 250 x g for 3. Wash cells once with 1X DPBS without calcium,
5 minutes at room temperature. Aspirate and discard magnesium, or phenol red.
the supernatant.
4. Aspirate DPBS and add StemPro Accutase reagent
8. Gently resuspend the cell pellet in pre-warmed complete to the cells, following the StemPro Accutase
astrocyte medium and remove a sample for counting. reagent instructions.

9. Determine the total number of cells and percent viability 5. Incubate for 5–10 minutes at 36°C–38°C. Rock the cells
using your method of choice. If necessary, add warm every ~5 minutes and check under a microscope for
complete astrocyte medium to the cells to achieve the detachment and dissociation toward single cells.
desired cell concentration and recount the cells.
6. When the cells have detached, add an equal volume
10. Plate cells in an uncoated tissue-culture treated (1:1) of conditioned medium (from step 2) to slow the
flask, plate, or Petri dish at a seeding density of StemPro Accutase reagent activity.
2 x 104 cells/cm2.
7. Transfer the cells to a 15 or 50 mL tube.
11. Incubate cells at 37°C, 5% CO2, and 90% humidity, and
change growth medium every 4–5 days. 8. Rinse culture vessels with 1 mL of complete astrocyte
medium and add it to the tube.
12. Astrocytes are ready for experiments 2–3 weeks
after culturing. 9. Centrifuge the tube for 5 minutes at 250 x g.

10. Aspirate and discard the supernatant.

62
 Neural cell culture and differentiation

11. With a prewetted pipette, suspend the pellet in 2–3 mL to allow RNA synthesis. Note that feeders should be
warm complete astrocyte medium. plated and allowed to attach for at least 24 hours prior to
plating neurons.
12. Count the live cells using a method of choice.
1. Prepare dishes coated with 100 μg/mL poly-D-lysine.
13. To replate human astrocytes, remove a Geltrex matrix–
coated plate from 2°C to 8°C storage and warm to 2. Passage rat or human primary astrocytes as
room temperature for 1 hour. Tip slightly to aspirate the described above.
Geltrex matrix solution.
3. Seed astrocytes in complete astrocyte medium at high
Note: Do not allow the plate to dry out. density, roughly 4 x 104 cells/cm2. The correct seeding
density must be determined empirically for the chosen
Note: Rat astrocytes do not require Geltrex matrix– substrate and the requirements of the assay.
coated plates.
4. Twenty-four hours later, wash the cells twice with the
14. Dilute the astrocytes to the desired concentration unsupplemented Neurobasal Plus Medium component
in complete astrocyte medium. We recommend from the B-27 Plus Neuronal Culture System, and then
4 x 104 cells/cm2 (360,000 cells in 2–3 mL in one well replace with complete neuronal medium.
of a 6-well plate).
5. Seed neurons in complete Neurobasal Plus Medium at
15. Immediately seed the cells on Geltrex-coated plates and the desired density for downstream applications.
incubate at 37°C, 5% CO2 in air, and 90% humidity.
6. Two to three days after seeding neurons, change half
16. Change the medium every 2 days with fresh complete of the medium with complete Neurobasal Plus Medium
astrocyte medium. containing 10–50 μM FUdR and 50 μM uridine.

7. After 3–4 days, perform a half-volume change with

Plating primary cortical astrocytes for fresh Neurobasal Plus Medium without FUdR or uridine.
support of neurons Continue feeding cells with half-volume changes
2–3 times per week.
Astrocytes are often grown as feeders to provide
metabolic, trophic, or synaptic support for neurons. Such
References
co-cultures are typically treated with anti-mitotic drugs
Lepore AC, Rauck B, Dejea C, et al. (2008) Focal transplantation-based astrocyte replacement is
to prevent overgrowth of the glia. Although cytosine neuroprotective in a model of motor neuron disease. Nat Neurosci 11:1294–1301.
arabinoside (AraC) is a commonly used anti-mitotic, it Maragakis NJ and Rothstein JD (2006) Mechanisms of disease: Astrocytes in neurodegenerative
can be toxic to neurons (Wallace and Johnson, 1989). disease. Nat Clin Pract Neurol 2:679–689.
Fluorodeoxyuridine (FUdR) is less toxic, but it is still Martin DP, Wallace TL, and Johnson EM Jr. (1990) Cytosine arabinoside kills postmitotic neurons
important to choose the lowest dose that is effective in a fashion resembling trophic factor deprivation: evidence that a deoxycytidine-dependent
process may be required for nerve growth factor signal transduction. J Neurosci 10:184–93.
in arresting proliferation. This is often in the range of
Pekny M and Nilsson M (2005) Astrocyte activation and reactive gliosis. Glia 50:427–434.
10–50 µM. Uridine (50 μM) is typically given as well

63
Neural cell culture and differentiation

Rothstein JD, Martin L, Levey AI, et al. (1994) Localization of neuronal and glial glutamate
transporters. Neuron 13:713–725.

Rothstein JD, Dykes-Hoberg M, Pardo CA, et al. (1996) Knockout of glutamate transporters
reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.
Neuron 16:675–686.

Schutte RJ, Xie Y, Ng NN, et al. (2018) Astrocyte-enriched feeder layers from cryopreserved
cells support differentiation of spontaneously active networks of human iPSC-derived neurons.
J Neurosci Methods 294:91–101.

Takano T, Oberheim N, Cotrina ML, and Nedergaard M (2009) Astrocytes and ischemic injury.
Stroke 40:S8–S12.

Wallace TL and Johnson EM Jr. (1989) Cytosine arabinoside kills postmitotic neurons: evidence
that deoxycytidine may have a role in neuronal survival that is independent of DNA synthesis.
J Neurosci 9:115–24.

Xu L, Sapolsky RM, and Giffard RG (2001) Differential sensitivity of murine astrocytes and
neurons from different brain regions to injury. Exp Neurol 169:416–424.

Zhao G and Flavin MP (2000) Differential sensitivity of rat hippocampal and cortical astrocytes to
oxygen-glucose deprivation injury. Neurosci Lett 285:177–180.

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 Neural cell culture and differentiation

14 Cryopreserving neural stem cells

Summary

There are numerous protocols available for cryopreserving Cryopreservation and recovery solution options:
neural stem cells (NSCs) derived from human embryonic
• Gibco™ PSC Cryopreservation Kit (Cat. No. A2644601):
stem cells. The primary objectives of these methods are
Includes PSC Cryomedium and RevitaCell™
the recovery of the cells post-thaw and the retention of
Supplement (100X), or Gibco™ CTS™ Synth-a-Freeze™
their multipotent properties. This chapter describes a
Medium (Cat. No. A1371301)
standardized cryopreservation protocol that optimizes
survival of NSCs post-thaw, while maintaining sublineage
differentiation capacity of the preserved cells. Tools and equipment
• Sterile 15 mL conical tubes
View this protocol online and order products at
• Tabletop centrifuge
thermofisher.com/neuroprotocol/cryo
• Thermo Scientific™ Nalgene™ General Long-Term
Storage Cryogenic Tubes (Cat. No. 5000-1020)
Required materials
• Thermo Scientific™ Mr. Frosty™ Freezing Container
Cells (Cat. No. 5100-0001)
• Neural stem cells (NSCs)
• 37°C water bath

Media and reagents

• Gibco™ StemPro™ NSC SFM (Cat. No. A1050901: This
kit contains KnockOut™ DMEM/F-12 Basal Medium
stored at 4°C; StemPro™ NSC SFM Supplement stored
at –20°C to –5°C in the dark; and bFGF Recombinant
Human and EGF Recombinant Human proteins stored at
4°C, desiccated.)

• Gibco™ GlutaMAX™ Supplement (Cat. No. 35050061)

• Optional: Gibco™ Antibiotic-Antimycotic (100X)

(Cat. No. 15240062)

• Gibco™ TrypLE™ Select Enzyme (1X), no phenol red

(Cat. No. 12563011)

• Gibco™ DPBS, no calcium, no magnesium

(Cat. No. 14190144)

• 100% isopropyl alcohol

• Gibco™ Geltrex™ LDEV-Free, hESC-Qualified,

Reduced Growth Factor Basement Membrane Matrix
(Cat. No. A1413301)
65
Neural cell culture and differentiation

Preparing media

Complete StemPro NSC SFM Freezing and recovery media

Complete StemPro NSC SFM consists of KnockOut Xeno-free PSC Cryomedium (a component in
DMEM/F-12 with StemPro NSC SFM Supplement, EGF, Cat. No. A26444601) and animal origin–free
and bFGF, combined with GlutaMAX Supplement. CTS Synth-a-Freeze Medium (Cat. No. A1371301) have
Complete medium is stable for 4 weeks when stored in shown utility for cryopreservation of NSCs. Once thawed,
the dark at 2°C to 8°C. these cryomedia are stable for up to 6 months when stored
at 2°C to 8°C protected from light.
To prepare 50 mL of complete StemPro NSC SFM,
aseptically mix the following components. For larger RevitaCell Supplement is provided as a 100X solution
volumes, increase the component amounts proportionally. for addition to growth medium (complete StemPro NSC
If desired, add 0.5 mL of Antibiotic-Antimycotic (100X) SFM) for the first 18–24 hours post-thaw to assist in
solution per 50 mL of complete medium. minimizing apoptosis and necrosis. RevitaCell Supplement
is a chemically defined recovery supplement containing
Component Final conc. Amount a specific ROCK inhibitor coupled with molecules that
KnockOut DMEM/F-12 1X 48.5 mL have antioxidant and free radical scavenger properties.
GlutaMAX Supplement 2 mM 0.5 mL Upon thaw, RevitaCell Supplement should be efficiently
bFGF 20 ng/mL 1 μg mixed by gentle inversion and dispensed into usage-size
EGF 20 ng/mL 1 μg aliquots. Aliquots should be stored frozen at –20°C to –5°C
StemPro NSC SFM 2% 1 mL protected from light. Avoid long-term (>5 days) storage at
Supplement* 2°C to 8°C.
* You may observe a white precipitate when thawing StemPro NSC SFM Supplement; this precipitate
will disappear when the supplement is completely thawed or dissolved.

66
 Neural cell culture and differentiation

Cryopreserving neural stem cells

Guidelines for cryopreserving neural stem cells StemPro NSC SFM immediately after the incubation
• Cryopreserve NSCs when they are 80–90% confluent period (according to the instructions below).
(2–4 days after seeding).
4. Detach the NSCs from the culture vessel by pipetting
• Freeze NSCs at a concentration of 1 x 106 to off the cells or by tapping the culture vessel against the
2.4 x 106 viable cells/mL and a volume of 1 mL/vial. heel of your hand.
• Xeno-free PSC Cryomedium or CTS Synth-a-Freeze
Medium can be used for cryopreservation of NSCs. 5. Quickly transfer the cell suspension to a 15 mL conical
Prechill cryomedium prior to use in cryopreservation tube containing the appropriate volume of complete
procedures to minimize toxicity of DMSO StemPro NSC SFM (see Table 14-1 on the next page) to
within these formulations. neutralize the TrypLE Select Enzyme.

• Do not incubate the NSCs in TrypLE Select Enzyme for 6. Mix the cell suspension by gentle inversion 3 times and
more than 5 minutes to avoid cell death. remove a small aliquot for assessment of cell count and
viability using the Invitrogen™ Countess™ II Automated
• Pre-label all cryovials and Mr. Frosty Freezing Containers
Cell Counter or traditional hemocytometer.
(filled with 250 mL of 100% isopropyl alcohol and
prechilled to 4°C) prior to addition of cells.
7. Centrifuge the NSCs at 200 x g for 5 minutes.

Freezing neural stem cells 8. Gently aspirate the medium, being careful to avoid the
1. When NSCs are 80–90% confluent (2–4 days after cell pellet, and add prechilled (4°C) freezing medium
seeding), aspirate the complete StemPro NSC SFM from dropwise to the cells while moving the conical tube
the culture vessel. back and forth; gently resuspend the cells to a final
concentration of 1 x 106 to 2.4 x 106 viable cells/mL.
2. Wash the cells once with DPBS, no calcium, no
magnesium (DPBS –/–), according to the volumes shown 9. Transfer 1 mL of the NSC suspension in freezing
in Table 14-1 on the next page. Aspirate the DPBS medium into each pre-labeled, prechilled (4°C) cryovial.
and discard.
10. Transfer the cryovials to the Mr. Frosty Freezing
3. Add room temperature TrypLE Select Enzyme, Container and place the container into a –80°C freezer.
according to the volumes shown in Table 14-1 on the This procedure ensures that the cells freeze slowly at
next page, to the culture vessel and incubate at 37°C for approximately –1°C/minute.
2–5 minutes.
11. The next day, transfer the cells into liquid nitrogen. Note
Note: Do not incubate the NSCs in TrypLE Select that it is important to avoid longer storage at –80°C.
Enzyme for more than 5 minutes to avoid cell death.
Neutralize TrypLE Select Enzyme by adding complete

67
Neural cell culture and differentiation

Table 14-1. Reagent volumes (per well or per dish). Recovery of cryopreserved neural stem cells
TrypLE StemPro NSC 1. Coat the culture vessels with the appropriate substrate
Culture vessel DPBS –/–
Select for Medium for on which to culture your NSCs. Recommended
(surface area) for wash
dissociation neutralization substrates include Geltrex LDEV-Free, hESC-Qualified,
6-well (10 cm2) 2 mL 1 mL 3 mL Reduced Growth Factor Basement Membrane Matrix
12-well (4 cm2) 1 mL 0.4 mL 1.2 mL (Cat. No. A1413301), Gibco™ CTS CELLstart™ Substrate
24-well (2 cm ) 2
0.5 mL 0.2 mL 0.6 mL (Cat. No. A1014201), or Gibco™ Laminin Mouse Protein,
35 mm (10 cm ) 2
2 mL 1 mL 3 mL Natural (Cat. No. 23017015). Aspirate coating matrix
60 mm (20 cm ) 2
4 mL 2 mL 6 mL immediately prior to seeding of recovered NSCs.
100 mm (60 cm ) 2
12 mL 6 mL 18 mL
Note: Do not allow matrices to dry out.

Guidelines for recovery of neural stem cells 2. Prepare recovery medium by supplementing complete
• Minimize the duration of exposure of NSCs to StemPro NSC SFM with RevitaCell Supplement at
cryomedium at 37°C by thawing until only a small ice 1X final concentration (e.g., add 100 μL RevitaCell
crystal remains. Avoid longer incubation at 37°C in Supplement to 10 mL of complete StemPro NSC SFM).
cryomedium solutions.
3. Quickly thaw NSCs in a 37°C water bath until a small
• Ensure dropwise addition of growth medium (complete ice crystal remains.
StemPro NSC SFM) to the NSCs in cryomedium to
avoid osmotic shock. 4. Transfer the vial to the laminar flow hood and disinfect it
• When using RevitaCell Supplement, do not include with 70% ethanol. Allow the ethanol to evaporate prior to
additional ROCK inhibitors such as Y-27632 or opening the vial.
Thiazovivin to the growth medium for recovery.
5. Gently triturate cells and transfer to a 15 mL
• Within 18–24 hours post-thaw, aspirate growth medium conical tube.
supplemented with RevitaCell Supplement and replenish
with fresh StemPro NSC SFM in the absence of 6. Add 5 mL of complete StemPro NSC SFM dropwise per
RevitaCell Supplement for the remainder of culture. 1 mL of cell suspension, while shaking the tube back
and forth to avoid osmotic shock.

7. Mix the cell suspension by gentle inversion 3 times.

8. Centrifuge the NSCs at 200 x g for 5 minutes. Aspirate

the supernatant and discard.

68
 Neural cell culture and differentiation

9. Resuspend the cell pellet in recovery medium A

(e.g., StemPro NSC SFM supplemented with 1X

RevitaCell Supplement) and perform cell count
using the Countess II Automated Cell Counter or
traditional hemocytometer.

10. Aspirate matrix solution from precoated plates and seed

NSCs at desired plating concentration for downstream
B
assay in recovery medium (e.g., StemPro NSC SFM
supplemented with 1X RevitaCell Supplement). For % viability direct post-thaw
100

NSCs an initial seeding density of ~20,000–50,000 Dead Cell Stain Negative)

(SYTOX AADvanced
75

viable cells/cm2 is recommended.

50

11. 18—24 hours post-seeding, aspirate recovery medium 25

and replace medium with complete StemPro NSC SFM.
Refresh medium every other day thereafter. 0
PSC Cryomedium CTS Synth-a-Freeze

Cryomedium

Expected results Figure 14-1. Direct post-thaw viability assessment of cryopreserved

H9 ESC-derived NSCs. Post-thaw viability was assessed by Invitrogen™
H9 ESC-derived NSCs cultured in complete StemPro NSC SYTOX™ AADvanced™ Dead Cell Stain Kit (Cat. No. S10349) using the
Invitrogen™ Attune™ NxT Flow Cytometer. Experiments for recommended
SFM were cryopreserved according to the instructions
cryomedia included (A) a gating strategy for cryopreservation and (B) a
provided above and evaluated for viability. While direct post-thaw viability percentage. The cryomedium is shown to provide
percentage viability direct post-thaw is commonly used >70% viability direct post-thaw of cryopreserved NSCs.
as a metric to assess the performance of a cryomedium
solution (Figure 14-1), following cryopreservation and
recovery of NSCs, additional cell death is not apparent
immediately post-thaw. Additional loss of cell viability
occurs over the first 24 hours post-thaw due to the
processes of apoptosis and necrosis (Baust et al.,
2000 and 2001) and is a direct reflection of the stress
on the NSCs during the cryopreservation and recovery
processes. To assess the impact of these processes,
NSCs were examined under a phase-contrast microscope
and assayed for viability (Figure 14-2). The cryomedium
is shown to provide >70% viability direct post-thaw of
cryopreserved NSCs, and RevitaCell Supplement was
shown to significantly improve post-thaw recovery of
NSCs cryopreserved in CTS Synth-a-Freeze Medium
or PSC Cryomedium.

69
 Neural cell culture and differentiation

A
PSC Cryomedium CTS Synth-a-Freeze Medium
+ RevitaCell Supplement + RevitaCell Supplement

B
70,000
PrestoBlue Cell Viability Reagent
24 hour post-thaw viability

60,000

50,000

40,000

30,000
CTS Synth-a-Freeze PSC Cryomedium
Cryomedium

Figure 14-2. 24-hour post-thaw viability assessment of

cryopreserved H9 ESC-derived NSCs. To assess 24-hour post-thaw
recovery, NSCs plated on Geltrex matrix–coated plates were examined
via (A) phase-contrast imaging and (B) Invitrogen™ PrestoBlue™ Cell
Viability Reagent (Cat. No. A13261). RevitaCell Supplement was shown to
significantly improve post-thaw recovery of NSCs cryopreserved in CTS
Synth-a-Freeze Medium or PSC Cryomedium; solid bars indicate recovery
in medium alone, while hatched bars indicate recovery in medium plus
RevitaCell Supplement.

70
 Neural cell culture and differentiation

Troubleshooting

For troubleshooting tips regarding cryopreservation and recovery of NSCs, see below.

Problem Possible cause Solution

• Ensure that NSCs are not overly confluent prior to cryopreservation;
target 80–90% confluency at time of harvest.
• Ensure that TrypLE Select Enzyme is not left on the NSCs for
>5 minutes.
• Ensure Mr. Frosty Freezing Container contains the appropriate
amount of 100% isopropyl alcohol. Be certain to refresh the
container with fresh isopropyl alcohol every 5 freeze cycles to ensure
proper cooling rate of about –1°C/minute.
NSCs stressed during
• Ensure that Mr. Frosty Freezing Container, cryovials, and
cryopreservation
cryomedium solutions are prechilled to 4°C prior to use.
• Ensure dropwise addition of prechilled cryomedia to the NSCs to
avoid osmotic shock.
• Limit the number of samples you are cryopreserving to minimize the
Low cell survival rate
toxicity of DMSO on your NSCs. Placing vials on ice can assist in
minimizing damage during the vialing process.
• Do not disturb the Mr. Frosty Freezing Container for up to 4 hours
post-placement in the –80°C freezer.
• Ensure that cells are not left at 37°C for extended periods of time. Be
certain to thaw NSCs until only a small ice crystal remains.
• Ensure dropwise addition of growth medium to the NSCs to avoid
NSCs stressed osmotic shock to the cells.
during recovery from • Limit the number of vials thawed at one time, as longer exposure of
cryopreservation cells to cryopreservation media can have a negative impact on post-
thaw viability.
• Ensure addition of RevitaCell Supplement to recovery medium for the
first 18–24 hours post-thaw to maximize cell survival.
Inconsistent cell • Always harvest cells at comparable confluency.
Non-reproducible confluency
cryopreservation and Inefficient mixing • Upon thaw, ensure efficient mixing of PSC Cryomedium,
recovery of cryomedium or CTS Synth-a-Freeze Medium, and RevitaCell Supplement by
RevitaCell Supplement gentle inversion.

References
Baust JM, VanBuskirk RG, Baust JG, et al. (2000) Cell viability improves following inhibition of Baust JM, Vogel MJ, VanBuskirk RG, et al. (2001) A molecular basis of cryopreservation failure
cryopreservation-induced apoptosis. In Vitro Cell Dev Biol Anim 36(4):262–270. and its modulation to improve cell survival. Cell Transplantation 10:561–571.

71
Neural cell culture and differentiation

72
Cell analysis
15 Cell viability assays for neurons and neural cells

Summary Required materials

The Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit Cells

provides a two-color fluorescence cell viability assay • Adherent or suspended NSCs or neurons
that is based on the simultaneous determination of live
and dead neurons and neural stem cells (NSCs) with Media and reagents
probes that measure two recognized parameters of • LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells
cell viability: intracellular esterase activity and plasma (Cat. No. L3224)
membrane integrity.
– Calcein AM
– Ethidium homodimer-1 (EthD-1)
The polyanionic dye calcein AM is well-retained within
live cells, producing intense uniform green fluorescence • Gibco™ DPBS, calcium, magnesium (Cat. No. 14040141)
in live cells (excitation/emission ~495 nm/~515 nm), while
ethidium homodimer-1 (EthD-1) enters cells with damaged Special tools
membranes to produce bright red fluorescence in dead • Fluorescence microscope
cells (excitation/emission ~495 nm/~635 nm).
• Invitrogen™ Attune™ NxT Flow Cytometer (if using
flow cytometry)
Protocols are provided for fluorescence microscopy or
microplate analysis of adherent cells, or flow cytometry
analysis of cells in suspension. Note: Calcein AM and EthD-1 can be viewed
simultaneously with a conventional fluorescein longpass
View this protocol online and order products at filter. The fluorescence from these dyes may also be
thermofisher.com/neuroprotocol/cellviability observed separately; calcein AM can be viewed with a
standard fluorescein bandpass filter, and EthD-1 can be
viewed with filters for propidium iodide or Texas Red™ dye.

73
Cell analysis

Preparing reagents Methods

Prepare the reagents in the LIVE/DEAD Viability/­ Determining the viability of adherent cells
Cytotoxicity Kit as follows: Adherent neurons or NSCs may be cultured on sterile glass
coverslips or in a multiwell plate. A protocol for a 96-well
1. Remove the stock solutions provided in the kit from the plate is described below.
freezer and allow them to warm to room temperature.
1. Aspirate the spent medium from the wells and rinse the
2. Add 20 μL of the supplied 2 mM EthD-1 stock solution cells gently with 200 μL of DPBS +/+ prior to the assay,
(Component B) to 10 mL of sterile, tissue culture– to remove or dilute any serum esterase activity.
grade DPBS, calcium, magnesium (DPBS +/+). Vortex
to ensure thorough mixing. This prepares a ~4 μM Note: Serum esterases could cause some increase in
EthD-1 solution. extracellular fluorescence by hydrolyzing calcein AM.

3. Combine the reagents by adding 5 μL of the supplied 2. Add 200 µL of working solution of LIVE/DEAD reagent.
4 mM calcein AM stock solution (Component A) to
the 10 mL of EthD-1 solution in DPBS +/+. Vortex the 3. Incubate for 30 minutes at 37°C.
resulting solution to ensure thorough mixing.
4. Rinse cells with 200 µL of DPBS +/+ prior to analyzing
Note: This reagent mixture is suitable for most neural cells either by fluorescence microscopy or by
cells. For cells with higher esterase activity, you might plate reader.
need to start with a lower calcein AM concentration.
For further information, refer to the user manual Determining viability of cells in suspension with flow
provided with the LIVE/DEAD Viability/Cytotoxicity Kit. cytometry using the Attune NxT Flow Cytometer
Allow all the reagents to come to room temperature
The resulting working solution of ~2 μM calcein AM before proceeding.
and ~4 μM EthD-1 is ready to be used. The final
concentration of DMSO is ≤0.1%, a level generally 1. Make an 80-fold dilution of calcein AM (Component A)
innocuous to most cells. in DMSO to make a 50 μM working solution (e.g., add
2 mL of calcein AM to 158 mL DMSO).
Note: Prepare a freshly coated culture vessel each
time before plating cells. There is no need to rinse the 2. Prepare a 1 mL suspension of cells with 0.1 x 106 to
culture vessel before use. 5 x 106 cells/mL for each assay. Cells may be in culture
medium or buffer.

3. Add 2 μL of a 50 μM calcein AM working solution and

4 μL of the 2 mM EthD-1 stock to each mL of cells.
Mix the sample.

74
 Cell analysis

4. Incubate the cells for 15–20 minutes at room Typical results

temperature, protected from light.

5. As soon as possible after the incubation period

(within 1–2 hours), analyze the stained cells by flow
cytometry using 488 nm excitation and measuring
green fluorescence emission for calcein AM
(i.e., 530/30 bandpass) and red fluorescence emission
for EthD-1 (i.e., 610/20 bandpass).

6. Gate on cells to exclude debris. Using single color–

stained cells, perform standard compensation. The
population should separate into two groups: live cells will
show green fluorescence and dead cells will show red
fluorescence (Figure 15-1).

Figure 15-1. Primary rat hippocampal neurons showing live (green)

and dead (red) cells using the LIVE/DEAD Viability/Cytotoxicity Kit.

Figure 15-2. Flow cytometry viability assay using the LIVE/DEAD

Viability/Cytotoxicity Kit. A 1:1 mixture of live and ethanol-fixed human
B cells was stained with calcein AM and EthD-1 following the protocol
provided. Flow cytometry analysis was performed with excitation at
488 nm. The resulting bivariate frequency distribution shows the clear
separation of the green fluorescent (530 nm) live cell population from the
red fluorescent (585 nm) dead cell population.

75
Cell analysis

16 Markers for characterizing neural subtypes

Summary

After cells are isolated from tissue or differentiated from View this protocol online and order products at
pluripotent precursors, the resulting population needs to thermofisher.com/neuroprotocol/markers
be characterized to confirm whether the target population
has been obtained. This chapter lists cell type–specific
antibody markers commonly used for immunocytochemical
(ICC) and flow cytometric analysis of neural subtypes.

Cell type–specific antibodies for characterizing neural subtypes

Cell type Antigen Antibody Cat. No.
Sox1 PA5-23351
Sox2 PA1-094, PA5-85144
Neural stem cells Nestin MA1-110
CD133 PA5-38014
Pax6 42-6600, MA1-109
MAP2 MA5-12823, 13-1500, MA5-12826
HuC/D A21271, A21272
Neurofilament MA1-2010, 34-1000, 13-0400, 13-1300
Neuronal progenitors H, M, L
Neurons (pan)
NCAM 701379, MA5-11563, PA5-78402
βIII tubulin MA1-118, 480011
Dcx 48-1200
TH P21962, 701949
Nurr1 MA1-195
Dopaminergic neurons
OTX2 701948, MA5-15854
LMX1a 710980
ISL1 MA5-15515, MA5-15516, PA5-27789
Motoneurons
HB9 PA5-23407
GABA PA5-32241
GABAergic neurons
GAD2/GAD65 PA5-22260
vGLUT1 48-2400
vGLUT2 MA5-27613
Glutaminergic neurons
NMDAR1 32-0500, PA3-102
NMDAR2B 71-8600, PA5-85362, PA3-104
Cholinergic neurons ChAT PA1-4738, PA5-29653

76
 Cell analysis

Cell type Antigen Antibody Cat. No.

CD44 701406, MA5-13887, MA5-13890, PA5-21419
GFAP MA5-12023, PA1-10019, PA1-10004, PA5-16291
Astrocyte progenitors
Astrocytes S100beta PA5-78161
Glutamine 710963
synthetase
GalC PA5-42652, PA5-27352
NG2 37-2300, 37-2700, 41-2000
Oligodendrocyte progenitors A2B5 MA1-90445
Oligodendrocytes Olig2 MA5-15810
MBP PA5-78397, PA1-10008
Olig1 PA5-21613, MA5-23954, MA5-22956, PA5-80870
IBA1 711504, MA5-27726, PA5-27436
CD11b 14-0112-82, 14-0118-82, CD11b00
CD68 MA5-13324, 11-0689-42
Microglia
HLA-DR 11-9956-42
CX3CR1 702321, 711353, PA5-19910
TREM-2 PA5-46980, 702886

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Cell analysis

17 Flow cytometry analysis

Summary Reagents and equipment

• 0.1% BSA in buffer (e.g., PBS, HBSS) or cell culture
Flow cytometry is a cell analysis application that allows medium (staining buffer)
for single-cell multiparametric quantitative analysis of
• Antibodies (primary and secondary, as needed
millions of cells in a short time. This provides a more
based on experiment)
comprehensive systemic analysis and understanding.
Flow cytometry accomplishes this by using lasers of • Cell health dye reagents (e.g., Invitrogen™
multiple wavelengths to excite fluorescent molecules. LIVE/DEAD™ reagents)
These fluorescent molecules can be antibody conjugates,
fluorescent cellular dyes, and fluorescent proteins. The • Flow cytometer (e.g., Invitrogen™ Attune™ NxT Flow
laser excites these fluorescent molecules to emit signals of Cytometer with acoustic focusing technology)
varying wavelengths that are further filtered by wavelength
and captured for quantitation. This ultimately allows for
a more comprehensive surveying of the expression and Titrating antibodies
density of particular proteins, nucleic acids, and other cell
characteristics of a cell population. Flow cytometry allows Determining the optimal concentration of antibody
for flexibility of interrogation, from the simple screening for flow cytometry
of cells transfected with fluorescent reporter proteins all 1. Dilute labeled antibodies for the appropriate antigens
the way to multiparametric analysis of different cellular to be detected in staining buffer. Make dilutions of all
traits such as cell health, metabolism, DNA changes, antibodies: undiluted, and 2-, 5-, 10-, 20-, 40-, 80-, and
cytokine formation, and other cell phenotypes, in a large 100-fold dilutions.
heterogeneous cell population of millions of cells.
2. Prepare the cells that express the antigen to
View this protocol online and order products at be analyzed.
thermofisher.com/neuroprotocol/flow
3. Count the number of cells.

Required materials 4. Use 1 x 106 cells for each dilution. Smaller numbers of
cells ranging from 50,000 to 100,000 may work as well.
Cells
• Cells in suspension 5. Centrifuge cells at 300 x g for 5 minutes at 4°C and
discard the supernatant.
• Adherent cells, trypsinized and in suspension buffer

• Dissociated tissue cells 6. Add 5 μL of antibody from each dilution into separate
sample tubes containing cells.

7. Prepare negative controls of cells that have not been

stained with antibody. If desired, cells labeled with an
isotype control can be used.

78
 Cell analysis

8. Mix well and incubate cells for 25–30 minutes. 6. Optional: Filter the cell suspension through a fine
mesh filter before analysis or sorting the cells by
9. Wash with 3 mL of staining buffer. Discard the flow cytometry.
supernatant and resuspend the cells in 0.5 mL of
staining buffer.
Two-step staining with
10. Analyze the cells by flow cytometry. biotinylated antibodies
Note: Use the same cell number in every experiment. Starting 1. For adherent cells, trypsinize and add staining buffer.
with larger numbers of cells is preferred since setting up Transfer the cells to a conical tube and centrifuge at
parameters during flow cytometry analysis takes time and 300 x g for 5 minutes. Discard the supernatant.
collecting >10,000 events produces more reliable data.
2. Add 5 μL of appropriately diluted biotinylated
Note: We recommended that you always use a dead cell primary antibody.
stain to identify dead cells in any immunophenotyping
experiment, as dead calls may nonspecifically bind 3. Resuspend the cell pellet by gently mixing and incubate
antibodies and give false readings. for 25–30 minutes.

4. Wash the cells with 3 mL staining buffer. Centrifuge the

One-step staining with fluorescent cells at 300 x g for 5 minutes.
labeled antibodies
5. Discard the supernatant. Add diluted streptavidin
1. For adherent cells, trypsinize and then add staining conjugated to a fluorescent tag.
buffer. Transfer the cells to a conical tube and centrifuge
at 300 x g for 5 minutes. Discard the supernatant. 6. Mix well and incubate the cells for 25–30 minutes.

2. Add the appropriate amount of diluted fluorescent 7. Wash the cells with 3 mL staining buffer. Centrifuge the
conjugated primary antibodies to the cell pellet. cells at 300 x g, 4°C for 5 minutes.

3. Resuspend the cell pellet by gentle mixing and incubate 8. Discard the supernatant and resuspend cells with
for 25–30 minutes. 0.5 mL of staining buffer.

4. Wash the cells with 3 mL staining buffer. Centrifuge the 9. Filter the cell suspension through a fine mesh filter
cells at 300 x g for 5 minutes. before analysis or sorting the cells by flow cytometry.

5. Discard the supernatant and resuspend the cells with

0.5 mL staining buffer.

79
Cell analysis

18 Immunocytochemistry

Summary Required materials

Immunocytochemistry is a technique used to assess Cells

the presence of a specific protein or antigen in cells by • Gibco™ Primary Rat Cortex Neurons (Cat. No. A1084001)
use of a specific antibody that binds to it. The antibody or Gibco™ Primary Rat Hippocampal Neurons
allows visualization of the protein under a microscope. (Cat. No. A1084101)
Immunocytochemistry is a valuable tool to study the
presence and subcellular localization of proteins. Media and reagents
• Gibco™ DPBS, calcium, magnesium (Cat. No. 14040141)
View this protocol online and order products at
• Gibco™ Goat Serum, New Zealand Origin
thermofisher.com/neuroprotocol/immuno
(Cat. No. 16210064)

• Primary antibodies to proteins of interest

• Secondary antibodies corresponding to primary antibody

source species

• Invitrogen™ 4´,6-Diamidino-2-Phenylindole,
Dihydrochloride (DAPI) (Cat. No. D1306) or Invitrogen™
NucBlue™ Fixed Cell ReadyProbes™ Reagent
(Cat. No. R37606)

• Invitrogen™ ProLong™ Gold Antifade Mountant

(Cat. No. P36930)

• Paraformaldehyde (4%) in DBPS

• Triton™ X-100 surfactant

Special tools
• Multi-chambered slides

• Fluorescence microscope

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 Cell analysis

Cell type–specific antibodies for characterizing neural subtypes

Cell type Antigen Antibody Cat. No. Source Reactivity Dilution Notes
Sox2 MA1-014 Mouse IgG2a Hu 1:200 Fig. 18-2
MA1-110 Rabbit Hu, Rt, Ms 1:100 Fig. 18-4
Neural stem cells Nestin
PA5-11887 Rabbit Hu 1:200
Pax6 42-6600 Rabbit Hu, Rt, Ms 1:200
13-1500 Mouse IgG1 Hu, Rt, Ms 1:400 Fig. 18-3
MAP2
PA5-17646 Rabbit Hu, Rt, Ms 1:250 Fig. 18-1
Neuronal progenitors HuC/D A21271 Mouse IgG2b Hu, Rt, Ms 1:200
Neurons (pan) Neurofilament MA5-14981 Rabbit Hu, Rt, Ms 1:100
Beta III tubulin 480011 Mouse IgG Hu, Rt, Ms 1:1,000
Dcx 48-1200 Rabbit Hu, Rt, Ms 1:400 Fig. 18-2
Dopaminergic neurons TH P21962 Rabbit Hu 1:1,000
GABA PA5-32241 Rabbit 1:5,000
GABAergic neurons
GAD2/GAD65 39-8200 Mouse IgG1 Hu, Rt, Ms 1:100 Fig. 18-3
vGLUT1 48-2400 Rabbit Hu, Rt, Ms 1:500 Fig. 18-3
Glutamatergic neurons vGLUT2 42-7800 Rabbit Hu, Rt, Ms 1:100
NMDAR1 32-0500 Mouse IgG2a Hu, Rt, Ms 1:100 Fig. 18-3
A25528 Mouse IgG Hu 1:50
CD44
Astrocyte progenitors MA5-13890 Mouse IgG2a Hu 1:1,000 Fig. 18-4
Astrocytes PA1-10019 Rabbit Hu, Rt, Ms 1:1,000
GFAP
13-0300 Rat Hu, Rt, Ms Fig. 18-1
Oligodendrocyte GalC Millipore MAB345 Mouse IgG Hu, Rt, Ms 1:200 Fig. 18-2
progenitors 43-3110 Mouse IgM Hu, Rt, Ms 1:100
Oligodendrocytes A2B5
MA1-90445 Mouse IgM Hu, Rt, Ms 1:100 Fig. 18-4
Proliferation Ki67 14-5698-82 Rat Hu, Rt, Ms 1:100

81
Cell analysis

Secondary antibodies
Ex/Em* (color) Alexa Fluor Dye No. Host Reactivity Cat. No. Concentration
Goat Mouse IgM A31552 1:1,000
Goat Mouse IgG A21049 1:1,000
346/442 (blue) 350 Goat Rat IgG A21093 1:1,000
Goat Rabbit IgG A21068 1:1,000
Donkey Goat IgG A21081 1:1,000
Goat Mouse IgM A21042 1:1,000
Goat Mouse IgG A11029 1:1,000
Goat Rat IgM A21212 1:1,000
495/519 (green) 488
Goat Rat IgG A11006 1:1,000
Goat Rabbit IgG A11034 1:1,000
Donkey Goat IgM A11055 1:1,000
Goat Mouse IgM A21044 1:1,000
Goat Mouse IgG A11032 1:1,000
Goat Rat IgM SA5-10012** 1:1,000
590/617 (red) 594**
Goat Rat IgG A11007 1:1,000
Goat Rabbit IgG A11037 1:1,000
Donkey Goat IgG A11058 1:1,000
Goat Mouse IgM M31504 1:500
496, 536, 565/576
NA Goat Mouse IgG P852 1:1,000
(red)
Goat Rabbit IgG P2771MP 1:1,000
* Approximate excitation and emission maxima, in nm; NA = not applicable.
** The Invitrogen™ Goat Anti-Rat IgM Cross-Adsorbed Secondary Antibody, DyLight™ 594 (Cat. No. SA5-10012) provides a high-intensity, photostable DyLight™ fluorescent replacement for the discontinued
Invitrogen™ Alexa Fluor™ 594 goat anti–rat IgM secondary antibody.

82
 Cell analysis

Methods

Immunocytochemistry analysis
1. Before proceeding, prepare a solution of 5% goat 9. Remove the solution from the wells and coat the cells
serum (or other serum appropriate for the secondary with primary antibody diluted in 5% serum solution.
antibodies) in DPBS, calcium, magnesium (DPBS +/+).
This solution will be used to coat the cells before 10. Incubate the coated cells at 2°C to 8°C overnight.
antibody detection and to dilute the antibody. Prepare
enough solution to completely coat the cells three times. 11. Rinse the cells 3 times with DPBS +/+.

2. When you are ready to perform the 12. Treat the cells with a secondary antibody diluted in 5%
immunocytochemistry procedure, aspirate the culture serum solution.
medium from each chamber and gently rinse the cells
twice with DPBS +/+, without dislodging the cells. 13. Incubate for 60 minutes at room temperature.

3. Treat the cells with 4% paraformaldehyde in PBS for 14. Rinse the cells 3 times with DPBS +/+.
20 minutes to fix them.
15. Stain the cells with a DAPI solution (3 ng/mL) for
4. Rinse the cells 3 times with DPBS +/+. 10 minutes.

5. Proceed to staining, or you may store samples for up to 16. Rinse the cells with DPBS +/+, and if desired, mount the
3–4 weeks in DPBS +/+ at 4°C. Do not allow cells to dry. cells with ProLong Gold Antifade Mountant. Observe the
cells under the microscope using filters that correspond
6. Permeabilize the cells with 0.3% Triton™ X-100 surfactant to the secondary antibody excitation/emission spectra.
(diluted in DPBS +/+) for 5 minutes at room temperature.

Note: If you are using a surface antigen such as

GalC, omit permeabilization step and do not include
Triton X-100 surfactant in the blocking buffer.

7. Rinse the cells 3 times with DPBS +/+.

8. Add enough 5% serum solution from step 1 to the

cells to coat them, and incubate for 60 minutes at
room temperature.

83
Cell analysis

Typical results
A B

C D

Figure 18-1. Primary rat hippocampal neurons. Immunofluorescence

detection of primary neuronal cells stained with mouse anti-MAP2 antibody
(green) and astrocytes stained with rabbit anti-GFAP antibody (red). Nuclei
are stained with DAPI (blue).
Figure 18-2. Fluorescence images (20x) of Gibco™ hNSCs that
have been cultured in complete Gibco™ StemPro™ NSC SFM for
three passages, and then allowed to differentiate into neurons,
oligodendrocytes, or astrocytes. Upon directed differentiation, cells
start to lose the undifferentiated NSC marker, nestin, but stain positive for
the differentiated cell–type markers Dcx, GalC, and GFAP. (A) Cells were
stained for the undifferentiated NSC markers nestin (red) and SOX2 (green)
prior to directed differentiation. Cells were then differentiated into neurons
and glial cells, and respectively stained (B) for the neuronal marker Dcx
(green), (C) for the oligodendrocyte marker GalC (red), or (D) for the
astrocyte marker, GFAP (green). Differentiation to (B) neurons and to (C)
oligodendrocytes was observed at day 7, and to (D) astrocytes at day 21.
The nuclei were counterstained with DAPI (blue) in panels B–D.

84
 Cell analysis

A B C

Figure 18-3. Fluorescence images of neurons cultured in Gibco™

Neurobasal™ Plus Medium supplemented with Gibco™ B27™ Plus
Supplement (50X). (A) Neurons stained with anti-GAD65 (green) and
anti-MAP2 (red), and (B) anti-VGLUT1 (green) and anti-MAP2 (red). (C)
Neurons stained with anti-NMDAR1. Nuclei were counterstained with DAPI
in all images.

A B C

Figure 18-4. Fluorescence images of precursor cells prior to

differentiation. (A) Rat glial precursor cells stained with anti-A2B5 (green).
(B) Human iPSC-derived neural stem cells stained with anti-CD44 (green).
(C) Human iPSC-derived neural stem cells stained with anti-nestin (green).
Nuclei were counterstained with DAPI in all images.

85
Cell analysis

19 Quantitative image analysis

Summary
Thermo Scientific™ HCS Studio™ Cell
Cellular imaging technology has long contributed to Analysis Software
significant advances in cell biology research. In fact, the
ability to visualize individual cells was a founding moment
in the field of cell biology. As technology has continued The Target Activation BioApplication is a general-
to develop, cellular imaging progressed from a useful purpose assay for intensity-based measurements
qualitative method to a powerful and essential quantitative of molecular localization with broad applicability
analysis tool for cell-based assays. Two technological across multiple disciplines. This assay calculates
developments that have synergistically assisted the measurements of fluorescent indicators of choice
emergence of quantitative image analysis are automated on a cell-by-cell basis in up to six channels, where
fluorescence microscopes and powerful image analysis a channel represents a fluorophore or a specific
software. The automation of microscopes has enabled exposure condition. An object mask is generated for
accurate and consistent exposure times and precise image channel 1 primary objects (e.g., nuclei, cell bodies,
acquisition, which are two critical factors for intensity- organelles), and is used to define the measurement
based comparisons of cellular markers. The development area for fluorescent intensity in downstream channels.
of user-friendly image analysis software with an array The software allows you to visualize object-intensity
of dynamic image analysis algorithms and informatics histograms for each image and enables you to set
tools enables scientists to make hundreds of parametric object intensity–based thresholds for each channel.
measurements and are adaptable to a broad range of cell Population and subpopulation analysis features allow
types and applications. Quantitative image analysis is now you to quantify the percentage of positively stained
an accurate and efficient method for cellular analysis. cells for each channel as well as subpopulations of
co-stained cells.
This convergence of technological advancements has
resulted in a new area of quantitative image analysis The Neuronal Profiling BioApplication enables
called high-content analysis (HCA) or high-content quantification of morphological changes in neurons,
screening (HCS). HCA can be described as a set of allowing control over selecting neurites based on
analytical methods using automated microscopy, multi- morphological as well as intensity differences.
parameter image processing, and visualization tools, to Selection of neurons is possible through the use of
extract quantitative data from cell populations. The high- nucleus, cell body, and neurite object identification
throughput and data-rich nature of HCA makes it a valuable parameters and identifying subpopulations
method for biological research, cell characterization, and of neurons through quantification of multiple
drug discovery studies. This chapter will present examples biological characteristics.
of implementing HCA for cell type characterization
and measurement of cell type purity along a neuronal
differentiation workflow. Examples of HCA data visualization
will also be illustrated.

86
 Cell analysis

Implementation of HCA for measuring

neuronal differentiation efficiency
The Gibco™ PSC Dopaminergic Neuron Differentiation
Kit (Cat. No. A3147701) enables the differentiation of
pluripotent stem cells (PSCs) to midbrain dopaminergic DAPI FOXA2 OTX2

neurons. The workflow for this kit is a three-step process Figure 19-2. FP progenitor cell characterization.
(Figure 19-1). hPSCs are first induced in Floor Plate
Specification Medium into midbrain-specified floor plate
progenitor (FP) cells. Next, FP cells are expanded as
adherent cultures in Floor Plate Cell Expansion Medium
and then cultured in suspension to form spheres. Finally,
the spheres are differentiated into mature dopaminergic
neurons in Dopaminergic Neuron Maturation Medium.

FOXA2/OTX2/DAPI FOXA2 TH/DAPI

TH + FOXA2

Specification Expand/bank Maturation Figure 19-3. Midbrain dopaminergic neurons.

(days 0–10) (days 11–20) (days 21–35)
The image acquisition and analysis presented in this
chapter was performed using the Thermo Scientific™
Figure 19-1. Simplified diagram of workflow from midbrain-specified
FP cells through expansion/banking to differentiated, mature
CellInsight™ CX5 High-Content Screening (HCS) Platform
dopaminergic neurons. (thermofisher.com/hcs). The CellInsight CX5 platform
includes the HCS Studio Cell Analysis Software,
To assess the efficiency of differentiation at key points along which contains over 20 pre-established and highly
this protocol, the Gibco™ Human Dopaminergic Neuron customizable assays.
Immunocytochemistry Kit (Cat. No. A29515) was used
for optimal image-based analysis of three key markers of
neural differentiation: OTX2 and FOXA2 for characterization
and quantification of FP progenitor cells (Figure 19-2),
and tyrosine hydroxylase (TH) for identification of matured
dopaminergic neurons (Figure 19-3).

87
Cell analysis

Quantification of percentage of FP cells

using HCA
The efficiency of the specification step can be measured by A
Raw image Object mask Object intensity histogram
quantifying the percentage of FP progenitor cells, using the
Target Activation image analysis assay. For this example, the
objective of the Target Activation assay is to measure the
percentage of cells that are expressing both FOXA2 and FOXA2
Real-time
visualization of
OTX2. Channel 1 DAPI-stained nuclei are used to generate positive and
negative cells

a primary object mask (Figure 19-4). Channels 2 and 3 are,

respectively, the FOXA2 and OTX2 stains where intensity
measurements will be performed. The primary object mask OTX2

is duplicated for channels 2 and 3, where average intensity B Subpopulation identification

measurements are made for each object. Intensity-based
thresholds are then set for each of channels 2 and 3.
Once the thresholds are set, the software can calculate Two-channel intensity
scatter plot
Cells positive for both
FOXA2 and OTX2

the percentage of co-stained cells. For the image set in

Figure 19-4, the Target Activation assay calculated that
92.3% of the cells are co-stained for FOXA2 and OTX2.
Figure 19-5 illustrates the data visualization features of the Figure 19-5. Object intensity threshold setting and subpopulation
HCS Studio software. Figure 19-5, panel A, demonstrates data visualization.

setting thresholds for each individual channel. Figure 19-5,

panel B, demonstrates the data visualization of the co-
stained subpopulation of cells.

Object identification Two-channel target activation

Ch 1 Ch2 Ch3

92.3% FP cells

DAPI FOXA2 OTX2

Object ID mask Intensity-based threshold set for each marker

Figure 19-4. Quantification of percentage of FP cells.

88
 Cell analysis

Quantification of percentage of
dopaminergic neurons using HCA
Upon completion of the maturation step, the percentage
of dopaminergic neurons can be quantified using the
Neuronal Profiling assay (Figure 19-6). In channel 1, a mask
is generated to identify and count DAPI-stained nuclei.
For channel 2, a mask is generated to identify and count
TH-positive cell bodies. The software can then calculate
the percentage of TH-positive cells for each image set. For
the image set in Figure 19-6, the neuronal profiling assay
calculated 31.3% dopaminergic neurons.

DAPI TH: 31.3% Composite

DAPI TH Composite

Nuclei identification mask Cell body identification mask

TH-positive cell body = 31.3%
Nuclei count = 805 TH-positive cell body = 252

Figure 19-6. Quantification of dopaminergic neurons using the

Neuronal Profiling assay.

89
Cell analysis

20 Intracellular calcium assay using fluo-4

Summary Preparing reagents

The following protocol describes how to perform Fluo-4 AM loading solution

fluo-4 dye–based measurements of cytosolic Fluo-4 AM loading solution consists of fluo-4 dye
calcium changes in neural stem cells in response to (Component A), PowerLoad™ Concentrate (Component
neurotransmitter applications. B), and probenecid (Component D) prepared in Live
Cell Imaging Solution (LCIS). An optional background
View this protocol online and order products at suppressor dye (Neuro Background Suppressor,
thermofisher.com/neuroprotocol/fluo4 Component C) is also included in the kit.

1. Prepare 100X probenecid by adding 1 mL of LCIS

Required materials to the probenecid vial (Component D). Freeze any
unused portions.
Cells
• Neural stem cells or primary neurons, cultured on poly-D- 2. Prepare 10 mL of fluo-4 AM loading solution by adding
lysine–coated 96-well plate or other culture vessel the following solutions to a 15 mL vial, in order:

Reagents a. Add 10 μL of fluo-4 AM Dye solution (Component A)

• Invitrogen™ Live Cell Imaging Solution (Cat. No. A14291DJ) to 100 μL of PowerLoad solution (Component B).
Mix well and add 8.7 mL of LCIS.
• Gibco™ Glucose Solution (Cat. No. A2494001)

• Invitrogen™ Fluo-4 Calcium Imaging Kit (Cat. No. F10489) b. Add 100 μL of 100X probenecid (to a final
concentration of 1X) and 100 μL of 200 g/L Glucose
– Fluo-4 AM dye in DMSO Solution (to a final glucose concentration of 10 mM).
– PowerLoad™ Concentrate (100X concentrate)
– Neuro Background Suppressor
c. Optional: Add 1 mL of 10X Neuro Background
– Probenecid (100X concentrate)
Suppressor (Component C). If the suppressor is not
• Neurotransmitters or ligands (e.g., acetylcholine, glutamate) used, add 1 mL of LCIS.

Tools and equipment Loading cells with fluo-4 AM loading solution in a

• Fluorescence inverted microscope 96-well plate
1. Wash the cells with 100 μL of LCIS.
• Invitrogen™ EVOS™ FL Imaging System or EVOS FL Auto
Imaging System (thermofisher.com/evos) 2. Load the cells with 100 μL of fluo-4 AM loading solution
• Invitrogen EVOS Light Cube, GFP (Cat. No. AMEP4651)
™ ™ per well of a 96-well plate. Adjust the volume as
appropriate for other culture vessels.

3. Incubate the cells for 30 minutes, and then at room

temperature for 30 minutes. Cells are ready for imaging.

90
 Cell analysis

If the Neuro Background Suppressor is not used, 1.75 Fluo-4 AM

remove the fluo-4 AM loading solution and replace with 1.50 Fluo-8 AM
LCIS containing 1X probenecid. 1.25
1.00

ΔF/F
0.75
Data analysis 0.50
0.25

1. Integrate the acquired fluo-4 520 nm emission signal for 0.00

0.00001 0.0001 0.001 0.01 0.1 1 10
each region of interest, normalize to the first ten data
Carbachol (µM)
points (F/F0), and then plot against time.
Figure 20-2. Comparison of fluo-4 and fluo-8. Similar EC50 values are
2. Set the response criteria. For example, an NSC might observed with fluo-4 and fluo-8 AM dyes for a dose response of carbachol
on M1 CHO-K1 cells, but the ΔF/F (signal to background) is higher for
be considered responsive to a given neurotransmitter
fluo-4 AM.
or ligand if the resulting normalized signal rises more
than 10% within 60 seconds following neurotransmitter
addition compared to the baseline signal. The number of
NSCs that exhibit clear changes in intracellular Ca2+
([Ca2+]i) depends on the neurotransmitter and
differentiation state of the NSCs.

A B

Figure 20-1. Detection of intracellular calcium using fluo-4 AM.

(A) Calcium waves in beating cardiomyocytes. (B) Calcium spikes
in firing neurons.

91
Cell analysis

21 Measuring membrane potential using the FluoVolt kit

Summary Preparing reagents

The Invitrogen™ FluoVolt™ membrane potential dye FluoVolt loading solution

represents the next generation in voltage-sensitive probes The protocol below provides instructions for performing the
and brings together the best characteristics of the fast- membrane potential assay using cells grown in a 35 mm
and slow-response membrane potential probes: it has a dish with 2 mL of culture medium.
sub-millisecond response time to changes in membrane
potential and displays a high magnitude of response. The To a 15 mL tube, add the following reagents in the order
following protocol describes how to perform FluoVolt dye– listed below to prepare fresh FluoVolt loading solution:
based measurements of changes in membrane potential.
Component Amount
View this protocol online and order products at 100X PowerLoad Concentrate 100 μL
thermofisher.com/order/catalog/product/F10488 FluoVolt dye, 1,000X 10 μL
Swirl to mix.
LCIS or physiological buffer of choice 10 mL
Required materials Optional: Glucose Solution (200 g/L) 100 μL
Invert the tube to mix.
Cells
• Neural stem cells, cultured on poly-D-lysine–coated
96-well plate or other culture vessel 10 mM glucose stock in LCIS
Dilute the stock Glucose Solution (200 g/L) 1:100 into
Media and reagents LCIS for a final glucose concentration of 10 mM. Keep
• Invitrogen™ FluoVolt™ Membrane Potential Kit this solution clean and free of contaminants to prevent
(Cat. No. F10488) bacterial, fungal, or yeast growth once glucose has
– FluoVolt Dye (1,000X concentrate) been added.
– PowerLoad™ Concentrate (100X)
– Neuro Background Suppressor (10X concentrate)

• Invitrogen™ Live Cell Imaging Solution (LCIS)

(Cat. No. A14291DJ)

• Invitrogen™ EVOS™ FL Imaging System or EVOS™ FL Auto

Imaging System (thermofisher.com/evos)

• Invitrogen™ Valinomycin (Cat. No. V1644)

• Optional: Gibco™ Glucose Solution (Cat. No. A2494001)

(for preparing 20 mM glucose stock in LCIS)

92
 Cell analysis

Loading NSCs with FluoVolt membrane Image cells loaded with FluoVolt dye
potential dye
Standard FITC settings should be used to visualize the
1. Remove medium from adherent cells and wash cells membrane staining of FluoVolt dye. Short exposures
twice in physiological buffer of choice or LCIS. (2 milliseconds or less) are possible with pixel
2 x 2 binning or greater, but will depend strongly on
2. Add 2 mL of FluoVolt loading solution (page 92) hardware configurations to measure rapid or successive
to cells, and incubate cells at room temperature for depolarizations. To confirm positive responses from the
15–30 minutes. dye, treat cells with 10 µM valinomycin (a potassium
ionophore) for 30 minutes, and then add an equal
3. Remove FluoVolt loading solution, and wash cells twice volume of isotonic potassium chloride (KCl) solution to
in physiological buffer of choice or LCIS. depolarize the cells.

4. Add 2 mL of physiological buffer of choice or LCIS. Cells Note: Isotonic KCl is composed of 140 mM KCl,
are now ready for live-cell imaging. 2.5 mM NaCl, 1.8 mM CaCl2, 1.0 mM MgCl2,
20 mM HEPES, 20 mM glucose and adjusted to
Optional: To suppress background fluorescence, add pH 7.4 with NaOH.
1:10 diluted Neuro Background Suppressor solution.
A B

Figure 21-1. Imaging of differentiated NG-108 cells loaded

with FluoVolt membrane potential dye. Cells were imaged with
10-millisecond illumination pulses and images acquired with 2 x 2 binning.
The three selected traces (A) show fluorogenic responses from the dye as
selected cells (B) spontaneously depolarize and repolarize in culture.

93
Cell analysis

A
25.0
di-8-ANEPPS
20.0

15.0

10.0
dF

5.0
B
0.0

-5.0 C
-10.0
Time (s)

25.0
FluoVolt membrane potential dye
20.0

15.0

B
10.0
dF

5.0

0.0

-5.0

-10.0
Time (s)

Figure 21-2. FluoVolt membrane potential dye vs. di-8-ANEPPS. (A)

Weaker signals, detected earlier by the FluoVolt membrane potential dye. A
series of 10 mV steps in voltage clamp from –100 to +30 mV. Changes as
small as 10 mV can be detected with the FluoVolt membrane potential dye.
(B) Greater magnitude of response displayed with the FluoVolt membrane
potential dye. (C) Unclamped control cell loaded with di-8-ANEPPS
shows photobleaching.

94
Molecular characterization
22 PCR primers for molecular characterization of
neural subtypes

Summary

After cells are isolated from tissue or differentiated from Over 1.8 million predesigned Applied Biosystems™
pluripotent precursors, the resulting populations should TaqMan® Gene Expression Assays covering more than 30
be characterized to confirm the presence of the desired species are available on our website (thermofisher.com/
population of cells. Further analysis can help define taqmangeneexpression). Alternatively, custom primers
molecular mechanisms underlying biological differences and arrays can be searched by disease, pathway, or
between different groups of cells (e.g., donors, treatments). biological process.
The table below lists PCR primers that can be used
in quantitative polymerase chain reactions (qPCR) to View this protocol online and order products at
measure the expression levels of specific genes for thermofisher.com/qpcr
characterizing neural stem cells (NSCs), mature neurons,
and neural subtypes as well as the neuronal supporting
cell types, including oligodendrocytes and astrocytes.

Target Gene Assay ID Amplicon size (bp)

SOX1 Hs01057642_s1 96
Neural stem cells SOX2 Hs01053049_s1 91
NES Hs04187831_g1 58
MAG Hs01114387_m1 74
Oligodendrocytes
OSP Hs00194440_m1 60
ALDH1L1 Hs01003842_m1 55
Astrocytes
GFAP Hs00909233_m1 57
MAP2 Hs00258900_m1 98
Neurons
CHAT Hs00758143_m1 65
Endogenous control ACTB Hs01060665_g1 63
GABAergic GAD1 Hs01065893_m1 100
Serotonergic neurons SLC6A4 Hs00984349_m1 58
Cholinergic neurons CHAT Hs00758143_m1 65
Dopaminergic neurons TH Hs00165941_m1 63

95
Molecular characterization

23 RNA isolation and cDNA preparation from neural cells

• Invitrogen™ SuperScript™ IV First-Strand Synthesis System

Summary
(Cat. No. 18091050)

A rapid method of analysis for determining the identity of – SuperScript IV Reverse Transcriptase
neural stem cells (NSCs), mature neurons (and specific – 5X SSIV Buffer Mix
subtypes), and glial cells involves the early detection of – 10 mM dNTP Mix
differentiation and lineage-specific markers tracked at the – 0.1 M DTT
RNA level. This protocol follows methodologies described – 50 μM oligo(dT)20
in the Invitrogen™ PureLink™ RNA Mini Kit manual for – Random hexamers (50 ng/µL)
isolating total RNA from neuronal cell types, followed by – Ribonuclease inhibitor
cDNA synthesis using Invitrogen™ SuperScript™ IV Reverse – Ribonuclease H (RNase H)
Transcriptase. The following protocol gives you a step-
• 70% ethanol
by-step procedure for template preparation required for
RT-PCR or RT-qPCR. • Gibco™ TrypLE™ Express Enzyme (1X), no phenol red
(Cat. No. 12604013)
View this protocol online and order products at
thermofisher.com/neuroprotocol/rna • Gibco™ DPBS, no calcium, no magnesium
(Cat. No. 14190144)

• Invitrogen™ Ribonuclease H (RNase H) (Cat. No. 18021071)

Required materials
• Invitrogen™ BlueJuice™ Gel Loading Buffer (10X)
Cells (Cat. No. 10816015)
• Neuronal cell types of interest
• Tabletop centrifuge
Reagents and equipment
• DEPC-treated water
• PureLink RNA Mini Kit (Cat. No. 12183018A)

– RNA Lysis Buffer RNA isolation

– Wash Buffer I
– Wash Buffer II Isolating RNA
– RNase-free water Important: Perform all steps on ice unless noted otherwise.
– RNA spin cartridges For all incubations, heat the thermocyclers in advance.
– Collection tubes Prechill all reagents and thaw all frozen reagents and cells
– RNA recovery tubes immediately prior to use. To prevent RNase contamination,
• Gibco™ 2-Mercaptoethanol (Cat. No. 21985023) wear disposable gloves while handling all materials and use
of sterile disposable plasticware and RNase-free pipette
tips with aerosol barriers is recommended. Always wear
appropriate personal protective equipment when working in
a laboratory environment.

96
 Molecular characterization

1. Prepare RNA Lysis Solution by adding 10 μL 10. Add 700 μL Wash Buffer I to the spin cartridge and
2-mercaptoethanol per mL of RNA Lysis Solution. centrifuge at room temperature for 15–30 seconds at
12,000 x g. Discard the flow-through and the tube. Place
2. Remove the medium from T-25 flasks, rinse once with the spin cartridge into a clean 2 mL RNA Wash Tube.
DPBS, no calcium, no magnesium (DPBS –/–), and treat
cells with 1 mL of pre-warmed TrypLE Express Enzyme 11. Add 500 μL Wash Buffer II (containing ethanol) to the
for 10 minutes at 37°C. spin cartridge and centrifuge at room temperature for
15–30 seconds at 12,000 x g. Discard the flow-through.
3. Harvest the cells and place them into 15 mL centrifuge
tubes. Take 100 μL of the sample and obtain a 12. Repeat step 11.
viable cell count.
13. Centrifuge for 1 minute to dry the cartridge.
4. Centrifuge the cells in a tabletop centrifuge for 7 minutes
at 100 x g. Discard the supernatant. 14. Place the cartridge into a clean RNA Recovery Tube.
Add 40 μL of RNase-free water to the cartridge,
5. Freeze the cells overnight in a –80°C freezer; frozen and let it stand for 1 minute. Centrifuge the cartridge
cell pellets can be stored at least a month if desired. at room temperature for 2 minutes at 12,000 x g.
Alternatively, freshly isolated cell pellets can be Add an additional 40 μL of RNase-free water to the
processed directly by going to step 6. cartridge and repeat the step. Yield should be about
60–300 μg total RNA.
6. Allow the cell pellet to thaw. Add 0.5 mL of RNA Lysis
Solution for each T-25 flask harvested for the pellet Note: Always allow time for the RNase-free water
(0.5 mL per 2 x 106 to 5 x 106 cells). Pipet the cells to percolate into the cartridge bed. Do not spin the
~20 times until the pellet is disrupted. cartridge immediately because it may result in partial
recovery and alter the yield of RNA.
7. Transfer 0.5 mL of cell lysis solution to 1.5 mL RNase-
free microcentrifuge tubes and centrifuge at room Determining RNA quality
temperature for 2 minutes at 12,000 x g. 1. Measure ratio of absorbance at 260 nm and
280 nm by analyzing 1 μL of the RNA sample using
8. Add 0.5 mL (1 volume) of 70% ethanol to each tube, and a Thermo Scientific™ NanoDrop™ spectrophotometer.
vortex the suspension 5–10 times. Conduct readings 3 times, and use the average as
the final value. Wipe down the analysis stage with a
9. Apply a 600 μL aliquot of sample to the RNA Spin lab tissue wetted with DEPC-treated water before and
Cartridge. Centrifuge at room temperature for after measuring each RNA sample. The A260/A280 of
15–30 seconds at 12,000 x g, then discard the flow- pure RNA is ~2.
through. Continue applying 600 μL aliquots of the same
RNA sample to the spin cartridge until the entire sample Note: The yield and quality of the isolated RNA
has been processed. depends on the type and age of the starting material,
in addition to how the material was collected
and preserved.

97
Molecular characterization

2. Prepare the RNA samples for RNA gel analysis Component Amount
as follows: 10 mM dNTP Mix 1 μL
Random hexamers (50 ng/μL) 1 μL
Component Amount RNA (1 μg) x μL
RNA sample 1 μL DEPC-treated water to 13 μL
BlueJuice Gel Loading Buffer (10X) 1 μL
3. Incubate the reaction in the thermocycler at 65°C
DEPC-treated water 8 μL for 5 minutes, and then immediately place on ice for
at least 1 minute. Collect the contents of the tube
3. Mix the components and load the samples onto by brief centrifugation.
individual wells of an agarose gel. Use 10 μL of 0.1 kb
and 1 kb molecular weight markers to estimate the 4. Vortex and briefly centrifuge the 5X SSIV Buffer Mix.
molecular weight size of ribosomal RNA bands. Use
10 μL DEPC-treated water for empty wells. Run 5. Prepare the reverse transcriptase reaction mix in a
samples for 30 minutes, visualize the bands on a separate tube using the components listed below:
UV light box, capture the gel image, and perform
band intensity measurements.
Component Amount
5X SSIV Buffer Mix 4 μL
RNA storage
100 mM DTT 1 μL
Store RNA samples at –80°C or process them further for
cDNA synthesis. Ribonuclease Inhibitor 1 μL
SuperScript IV Reverse Transcriptase 1 μL

cDNA preparation 6. Cap the tube, mix, and then briefly centrifuge
the contents.
First-strand cDNA synthesis
This protocol follows the methodologies described 7. Add the reverse transcriptase reaction mix to the
in the instructions for the SuperScript IV First-Strand annealed RNA.
Synthesis System.
8. Incubate the tube at 50°C for 10 minutes.
1. Mix and briefly centrifuge each component before
use. Pre-heat the thermocycler to 65°C. Note that 9. Terminate reaction by incubating at 80°C for 10 minutes,
oligo(dT)20 may be substituted for random hexamers then chill the tube on ice.
primer solution.
10. Add 1 μL of RNase H to the sample, and incubate at
2. Combine the following components on ice in a 0.2 mL 37°C for 20 minutes.
thin-walled PCR tube. Use a volume containing up to
1 μg of total RNA for the reaction. 11. Store the cDNA samples at –20°C or proceed to
PCR amplification.

98
 Molecular characterization

24 Characterizing neural cells by qPCR

Summary Special tools

• 7300 Real-Time PCR System or similar instrument
Quantitative polymerase chain reaction (qPCR) is one of
• 96-well or 384-well PCR plates (check the adapter type
the most accurate and sensitive methods for studying gene
installed in your real-time PCR machine and choose a
regulation, and can be used to measure the expression
compatible plate format; for more information, refer to the
levels of specific genes in a wide variety of neuronal
TaqMan Gene Expression Master Mix protocol)
cell models. Understanding gene patterns can provide
significant insight into diverse biological processes, • Vortex mixer
including the regulation of differentiation and maturation,
the impact of different cell-to-cell interactions, and • Microcentrifuge for 1.5 mL tubes
mechanisms of disease and aging.
• Applied Biosystems™ MicroAmp™ Optical Adhesive Film
(Cat. No. 4311971)
Here we provide guidelines and a general protocol for
performing qPCR using the Applied Biosystems™ 7300
Real-Time PCR System and TaqMan® Gene Expression
Master Mix. Methods

View this protocol online and order products at Template preparation

thermofisher.com/neuroprotocol/qpcr For qPCR, prepare cDNA generated using the protocol
described in “RNA isolation and cDNA preparation from
neural cells” (on page 96).
Required materials
Real-time PCR instruments
Starting material TaqMan Gene Expression Master Mix can be used with
• cDNA generated from total RNA isolated from neural cells a variety of real-time PCR instruments, including but not
(see “RNA isolation and cDNA preparation from neural limited to the following Applied Biosystems™ instruments:
cells” on page 9796) 7300 and 7500 Real-Time PCR Systems; ABI PRISM™
7000, 7700, and 7900HT systems; and GeneAmp™ 5700
Media and reagents system. Prefixed cycling conditions will be used across
• TaqMan Gene Expression Master Mix (Cat. No. 4369016) instruments.

• Applied Biosystems™ TaqMan® Gene Expression Assay Note: Because TaqMan Gene Expression Master
(FAM) (Cat. No. 4331182; and see “PCR primers for Mix is not supported for use with Fast Mode thermal
molecular characterization of neural subtypes” on cycling conditions, select the Standard Mode thermal
page 95) cycling condition.
• Invitrogen™ Nuclease-Free Water (not DEPC-Treated)
(Cat. No. AM9938)

99
Molecular characterization

Prepare the PCR assay mix (genes) Prepare the PCR assay plate
We recommend performing four replicates of each 1. Transfer 19 μL of each reaction mixture to each well of
reaction. In this protocol, we describe conditions for a an optical plate.
20 μL reaction size. For more information, visit
thermofisher.com/qpcr 2. Transfer 1 μL of cDNA template to each well. Each well
will have a total volume of 20 μL for the amplification.
Component Amount
TaqMan Gene Expression Master Mix (2X) 10 μL 3. Cover the plate with a MicroAmp Optical Adhesive Film.
Taqman Gene Expression Assay 1 μL
Nuclease-Free Water (not DEPC-Treated) 8 μL 4. Centrifuge the plate briefly to spin down the contents
and eliminate air bubbles from the solutions.
Total volume 19 μL

• Calculate the volume of each component of the PCR Run the PCR assay
assay mix by multiplying the volume of each component 1. Place the reaction plate in the instrument.
by the number of replicates for each sample.
2. Confirm or select sample volume of 20 μL.
• Include excess volume for the loss that occurs during
reagent transfers. 3. Under the Thermal Cycler tab, select standard mode
specified in the following table.
• Use 10 to 100 ng of cDNA per replicate.

• Cap the tubes and vortex briefly to mix the solutions.

AmpliTaq
• Centrifuge the tubes briefly to spin down the contents UDG* Gold, UP
PCR
incubation enzyme
and eliminate any air bubbles from the solutions.
Step activation
Cycle (40 cycles)
Hold Hold Anneal/
Denature
extend
Time 2 min 10 min 15 sec 1 min
Temp 50°C 95°C 95°C 60°C
* UDG: uracil-DNA glycosylase.

Note: The 2-minute, 50°C step is required for optimal

UDG enzyme activity. The 10-minute, 95°C step is
required to activate the AmpliTaq Gold™, UP enzyme.

4. Start the run.

100
 Molecular characterization

Analyzing results a
b

Threshold c
The general process for analyzing the data from gene
expression assays involves: Rn

d
1. Viewing the amplification plots for the entire plate.

2. Setting the baseline and threshold values to determine 0 5 10 15 20 25 30 35 40

the threshold cycles (Ct) for the amplification curves. Cycle number
e

a. Plateau phase
b. Linear phase
Note: When using Applied Biosystems™ real-time PCR c. Exponential (geometric) phase
instruments, you can use the Sequence Detection d. Background
e. Baseline
System (SDS) software to either automatically calculate
or manually set the baseline and threshold for the Figure 24-1. Typical amplification curve.
amplification curves.

3. Using the relative standard curve method or the

comparative Ct method to analyze your data.

Note: After analysis, verify that the baseline and

threshold were called correctly for each well by viewing
the resulting amplification plots, and adjust the values
manually if necessary. For more information, refer to
the TaqMan Gene Expression Master Mix protocol.

101
102
Transfection
25 Lipid-mediated transfection of neuronal cells

Culture conditions

The following table summarizes the culture conditions for cells, refer to the instructions supplied with the specific cell
various Gibco™ neural cell lines, including neural stem cells. line you are using.
For detailed instructions on culturing and passaging these

Cell type Media Culture conditions

• Adherent culture on Gibco™ Geltrex™ matrix or
CELLstart™ CTS™ Substrate–, fibronectin-, or
Complete Gibco™ StemPro™ poly-L-ornithine–coated culture vessels
Human Neural Stem Cells
NSC SFM1
• 37°C, humidified atmosphere of 5% CO2 in air
• Exchange spent medium every other day
• Adherent culture on Geltrex matrix–coated tissue
culture vessels
Human Astrocytes Complete Gibco™ Astrocyte Medium1
• 37°C, humidified atmosphere of 5% CO2 in air
• Exchange spent medium every 3–4 days
• Adherent culture on Geltrex matrix or CELLstart
CTS Substrate–, fibronectin-, or poly-L-ornithine–
Rat Fetal Neural Stem Cells Complete StemPro NSC SFM1 coated culture vessels
• 37°C, humidified atmosphere of 5% CO2 in air
• Exchange spent medium every 3–4 days
• Adherent culture on standard culture vessels
Rat Primary Cortical
Complete Astrocyte Medium 1,2
• 37°C, humidified atmosphere of 5% CO2 in air
Astrocytes
• Exchange spent medium every 2–3 days
• Adherent culture on CELLstart CTS Substrate–
Complete StemPro NSC SFM1, or poly-L-ornithine–coated culture vessels
Rat Glial Precursor Cells supplemented with 10 ng/mL
• 37°C, humidified atmosphere of 5% CO2 in air
PDGF-AA
• Exchange spent medium every other day
1 See “Preparing media” on page 115 for instructions on preparing complete StemPro NSC SFM and complete Astrocyte Medium.
2 For increased proliferation of rat astrocytes, you can supplement complete Astrocyte Medium (DMEM with 1X N-2 Supplement and 10% One Shot™ FBS) with 20 ng/mL EGF. Adding EGF to human astrocyte
cultures can increase proliferation, but may result in morphological or phenotypic changes.

103
Transfection

Summary Best practices

The following protocols provide instructions for lipid- The following guidelines are meant to improve your
mediated transfection of plasmid DNA, siRNA, or mRNA transfection workflow and its subsequent results.
into various neural cells using Invitrogen™ Lipofectamine™
• While our standard protocol has been significantly
3000, Lipofectamine™ 2000, Lipofectamine™ Stem,
simplified to reduce the number of optimization
Lipofectamine™ RNAiMAX, and Lipofectamine™
parameters, we recommend that you use best practices
MessengerMAX™ Transfection Reagents.
to further optimize your specific experimental protocol.
• Lipofectamine 3000 reagent is a next-generation, broad-
• While not always necessary, if you use antibiotics during
spectrum reagent for the delivery of plasmid DNA into
transfection, test your conditions thoroughly. Note that
immortalized neural cells with low cytotoxicity.
adding antibiotics to media during transfection may result
• Lipofectamine 2000 reagent is a broad-spectrum, animal in cell death.
origin–free formulation recommended for the delivery of
• Maintain the same seeding conditions between
plasmid DNA into primary neurons with low cytotoxicity.
experiments. When using immortalized cell lines, try to
• Lipofectamine Stem reagent is a versatile transfection use post-thaw cells between passages 4 and 25; ensure
reagent optimized to deliver multiple payloads (small the cells are healthy, and are between 70–90% confluent
and large DNA plasmids, mRNA, and Cas9 protein before transfection. In some instances, certain cells
complexes) into stem cells, including neural stem cells. achieve better results at lower confluency. In addition, be
Visit thermofisher.com/lipofectaminestem to learn sure to maintain consistent confluency across each well.
more about stem cell transfection.
• Use Gibco™ Opti-MEM™ I Reduced Serum Medium to
• Lipofectamine RNAiMAX reagent is a leading RNAi dilute the payload (DNA, siRNA, or mRNA) and the
transfection reagent optimized for the transfection of appropriate transfection reagent. Transfections can be
siRNA and Invitrogen™ Stealth RNAi™ duplexes into all performed in either the presence or absence of serum.
neuronal-based cell types, immortalized and primary. If serum-free medium is required, be sure to test it for
compatibility with the transfection reagent.
• Lipofectamine MessengerMAX reagent is an mRNA
transfection reagent that offers an alternative strategy
to transfect astrocytes, neural stem cells, neurons,
and other primary cells. If you are not satisfied with the
results obtained transfecting plasmid DNA, we highly
recommend this as an alternative for superior transfection
results. Visit thermofisher.com/messengermax to learn
more about the benefits of mRNA transfection.

View this protocol online and order products at

thermofisher.com/transfection

104
 Transfection

Optimization: a how-to example

Optimizing your transfection experiment is one of the most The example below helps you plan initial optimization
important steps in achieving the best results. The final experiments using the standard transfection protocols as
results can be heavily influenced by the number of cells, the starting points. It varies reagent doses and incubation
cell confluency, payload quality, incubation time, culturing times, but allows for adjustment of other parameters,
medium, and other factors. including payload amounts, as needed.

Example
Plate format 6-well
Cells SH-SY5Y: 70% confluent
Payload Plasmid DNA: 2,500 ng Constant
Recommended reagent doses* Lipofectamine 3000 reagent: 3.75 µL/well and 7.5 µL/well Variable
Reagent 2 P3000 reagent: 5 µL/well Constant
Medium Opti-MEM I Reduced Serum Medium: 250 µL/well
™
Constant
Incubation time 2 days, 3 days, or 4 days at 37°C Variable

Lipofectamine 3000 3.75 µL/well pDNA pDNA pDNA

P3000 5 µL/well
2,500 ng 2,500 ng 2,500 ng
Opti-MEM I 250 µL/well

Day 2–4 incubation at 37°C

Lipofectamine 3000 7.5 µL/well
P3000 5 µL/well pDNA pDNA pDNA
Opti-MEM I 250 µL/well 2,500 ng 2,500 ng 2,500 ng

* The recommended doses for Lipofectamine 3000 reagent were identified through extensive testing for the plasmid DNA (pDNA) payload. It was determined that these two doses were the most effective
across dozens of cell types.

105
Transfection

Transfecting plasmid DNA (immortalized neuronal cells)—protocol

Materials needed
• Plasmid DNA of interest (0.5–5 μg/μL) • Gibco™ Opti-MEM™ I Reduced Serum Medium
(Cat. No. 31985062)
• Invitrogen™ Lipofectamine™ 3000 Transfection Reagent
(Cat. No. L3000008) • Microcentrifuge tubes

– Lipofectamine 3000 reagent

– P3000 reagent Note: The following protocol has two reagent doses (dose 1
and dose 2) that are needed for the initial optimization only.

6-well format*
Step Description Component
Dose 1/well Dose 2/well
1 Seed cells to be 70–90% confluent
Adherent cells 0.25–1 x 106 0.25–1 x 106
at transfection
2 Prepare reagent mix: Dilute Opti-MEM I medium 125 µL 125 µL
Lipofectamine 3000 reagent in
Opti-MEM I Reduced Serum Medium Lipofectamine 3000 reagent 3.75 µL 7.5 µL
2a Vortex reagent mix 2–3 seconds
3 Dilute DNA: Prepare master mix of DNA Opti-MEM I medium 125 µL 125 µL
by diluting DNA in Opti-MEM I medium, DNA 2.5 µg (2,500 ng) 2.5 µg (2,500 ng)
then add P3000 reagent
P3000 reagent (2 µL/µg DNA) 5 µL 5 µL
4 Add diluted DNA (step 3) to each tube of Reagent mix 125 µL 125 µL
reagent mix (step 2) in a 1:1 ratio Diluted DNA 125 µL 125 µL
4a Incubate DNA–reagent complex 10–15 minutes at
room temperature
5 Add DNA–reagent complex to cells
6 Incubate transfected cells Incubate cells for 2–4 days at 37°C
7 Analyze the transfected cells
* See the experimental scaling table below to adjust for other plate formats.

Experiment scaling
Culture Reagent mix 1 (step 2) Diluted DNA (step 3)
Confluency
vessel Dose 1 Dose 2 Opti-MEM I P3000 DNA Opti-MEM I
6 well 0.25–1 x 106 3.75 µL 7.5 µL 125 µL 5 µL 2.5 µg (2,500 ng) 125 µL
24 well 0.5–2 x 10 5
0.75 µL 1.5 µL 25 µL 1 µL 0.5 µg (500 ng) 25 µL
96 well 1–4 x 10 4
0.15 µL 0.3 µL 5 µL 0.2 µL 0.1 µg (100 ng) 5 µL

106
 Transfection

Transfecting plasmid DNA (primary neuronal cells)—protocol

Materials needed
• Plasmid DNA of interest (100 ng/μL or higher) • Microcentrifuge tubes

• Invitrogen™ Lipofectamine™ 2000 Transfection Reagent Note: The following protocol has four reagent doses
(Cat. No. 11668030) (dose 1–4) that are needed for the initial optimization only.
• Gibco™ Opti-MEM™ I Reduced Serum Medium
(Cat. No. 31985062)
6-well format*
Step Description Component
Dose 1/well Dose 2/well Dose 3/well Dose 4/well
1 Seed cells to be 70–90%
Adherent cells 0.25–1 x 106 0.25–1 x 106 0.25–1 x 106 0.25–1 x 106
confluent at transfection
2 Prepare reagent mix: Dilute Opti-MEM I
150 µL 150 µL 150 µL 150 µL
Lipofectamine 2000 reagent medium
in Opti-MEM I Reduced Lipofectamine
Serum Medium 6 µL 9 µL 12 µL 15 µL
2000 reagent
2a Vortex reagent mix 2–3 seconds
3 Dilute DNA in Opti-MEM I Opti-MEM I
150 µL 150 µL 150 µL 150 µL
Reduced Serum Medium medium
2.5 µg 2.5 µg 2.5 µg 2.5 µg
DNA
(2,500 ng) (2,500 ng) (2,500 ng) (2,500 ng)
4 Add diluted DNA (step 3) to each Reagent mix 150 µL 150 µL 150 µL 150 µL
tube of reagent mix (step 2) in a
1:1 ratio Dilute DNA 150 µL 150 µL 150 µL 150 µL
4a Incubate DNA–reagent complex 5 minutes at room temperature
5 Add DNA–reagent complex
to cells
6 Incubate transfected cells Incubate cells for 1–3 days at 37°C
7 Analyze the transfected cells
* See the experimental scaling table below to adjust for other plate formats.

Experiment scaling
Culture Reagent mix (step 2) Diluted DNA (step 3)
Confluency
vessel Dose 1 Dose 2 Dose 3 Dose 4 DNA Opti-MEM I
6 well 0.25–1 x 10 6
6 µL 9 µL 12 µL 15 µL 2.5 µg (2,500 ng) 150 µL
24 well 0.5–2 x 10 5
2 µL 3 µL 4 µL 5 µL 0.5 µg (500 ng) 50 µL
96 well 1–4 x 10 4
1 µL 1.5 µL 2 µL 2.5 µL 0.1 µg (100 ng) 25 µL

107
Transfection

Expected results

A B

Figure 25-1. Plasmid DNA delivered by Lipofectamine 2000 reagent in

primary cortical neurons cultured in the Gibco™ B-27™ Plus Neuronal
Culture System (freshly isolated E18 rat; 6 days in vitro). (A) GFP
expression and (B) brightfield images, analyzed 48 hours posttransfection.

108
 Transfection

Transfecting siRNA (immortalized neuronal cells and primary neuronal cells)—protocol

Materials needed
• Invitrogen™ Silencer™ Select siRNAs • Gibco™ Opti-MEM™ I Reduced Serum Medium
(thermofisher.com/sirna) (Cat. No. 31985062)

• Invitrogen™ Lipofectamine™ RNAiMAX™ Transfection • Microcentrifuge tubes

Reagent (Cat. No. 13778075)

6-well format*
Step Description Component
Dose/well
1 Seed cells to be 60–80% confluent
Adherent cells 0.25–1 x 106
at transfection
2 Prepare reagent mix: Dilute Lipofectamine Opti-MEM I medium 150 µL
RNAiMAX reagent in Opti-MEM I Reduced
Serum Medium in one microcentrifuge tube Lipofectamine RNAiMAX reagent 7.5 µL
2a Vortex reagent mix 2–3 seconds
3 Dilute Silencer Select siRNA in Opti-MEM I Opti-MEM I medium 150 µL
medium in one microcentrifuge tube siRNA (10 µM) 30 pmol
4 Add diluted siRNA (step 3) to each tube of Reagent mix 150 µL
reagent mix (step 2) in a 1:1 ratio Diluted siRNA 150 µL
4a Incubate siRNA–reagent complex 5 minutes at
room temperature
5 Add siRNA–reagent complex to cells
6 Incubate transfected cells Incubate cells for
1–3 days at 37°C
7 Analyze the transfected cells
* See the experimental scaling table below to adjust for other plate formats.

Experiment scaling
Culture Reagent mix (step 2) Diluted siRNA (step 3)
Confluency
vessel Dose Opti-MEM I siRNA Opti-MEM I
6 well 0.25–1 x 10 6
7.5 µL 150 µL 30 pmol 150 µL
24 well 0.5–2 x 10 5
1.5 µL 50 µL 5 pmol 50 µL
96 well 1–4 x 10 4
0.5 µL 25 µL 1 pmol 25 µL

109
Transfection

Transfecting mRNA (immortalized and primary neuronal cells)—protocol

Materials needed
• mRNA of interest (0.5–5 µg/µL) • Gibco™ Opti-MEM™ I Reduced Serum Medium
(Cat. No. 31985062)
• Invitrogen™ mMESSAGE mMACHINE™ T7 ULTRA
Transcription Kit (Cat. No. AM1345) • Microcentrifuge tubes

• Invitrogen™ Lipofectamine™ MessengerMAX™ Transfection

Reagent (Cat. No. LMRNA008) Note: The following protocol has two reagent doses (dose 1
and dose 2) that are needed for the initial optimization only.

6-well format*
Step Description Component
Dose 1/well Dose 2/well
1 Seed cells to be 70–90% confluent
Adherent cells 0.25–1 x 106 0.25–1 x 106
at transfection
2 Prepare reagent mix: Dilute Opti-MEM I medium 125 µL 125 µL
MessengerMAX reagent in Opti-MEM I
Reduced Serum Medium MessengerMAX reagent 3.75 µL 7.5 µL
2a Vortex reagent mix 2–3 seconds
2b Incubate reagent mix 10 minutes at room temperature
3 Dilute mRNA in Opti-MEM I medium Opti-MEM I medium 125 µL 125 µL
mRNA 2.5 µg (2,500 ng) 2.5 µg (2,500 ng)
4 Add diluted mRNA (step 3) to each tube of Reagent mix 125 µL 125 µL
reagent mix (step 2) in a 1:1 ratio Diluted mRNA 125 µL 125 µL
4a Incubate mRNA–reagent complex 5 minutes at room temperature
5 Add mRNA–reagent complex to cells
6 Incubate transfected cells Incubate cells for 2–4 days at 37°C
7 Analyze the transfected cells
* See the experimental scaling table below to adjust for other plate formats. Visit thermofisher.com/lipofectaminestem for additional information on setting up mRNA transfection in NSCs using Lipofectamine
Stem Transfection Reagent.

Experiment scaling
Culture Reagent mix 1 (step 2) Dilute mRNA (step 3)
Confluency
vessel Dose 1 Dose 2 Opti-MEM I mRNA Opti-MEM I
6 well 0.25–1 x 106 3.75 µL 7.5 µL 125 µL 2.5 µg (2,500 ng) 125 µL
24 well 0.5–2 x 10 5
0.75 µL 1.5 µL 25 µL 0.5 µg (500 ng) 25 µL
96 well 1–4 x 10 4
0.15 µL 0.3 µL 5 µL 0.1 µg (100 ng) 5 µL

110
 Transfection

Expected results
A GFP expression 62% B GFP expression 76% C GFP expression 92%

Figure 25-2. Lipofectamine MessengerMAX reagent was used

to deliver mRNA encoding GFP (500 ng/well). Cells used included
(A) primary cortical neurons (freshly isolated from E16 mouse; 5 days
in vitro), (B) SK-N-SH cells (neuroblastoma) in a 24-well format, and
(C) Gibco™ Human Neural Stem Cells (H9-Derived, Cat. No. N7800100)
(hNSCs; 250 ng/well of mRNA was used in a 48-well format). GFP
expression (noted above) was analyzed 24 hours posttransfection.

111
Transfection

Transfecting plasmid DNA (neural stem cells)—protocol*

Materials needed
• Invitrogen™ Lipofectamine™ Stem Reagent • Microcentrifuge tubes
(Cat. No. STEM00003)
• Gibco™ Opti-MEM™ I Reduced Serum Medium
• Gibco™ StemPro™ NSC SFM (Cat. No. A1050901) (Cat. No. 31985062)

24-well format*
Step Description Component Dose 1/ Dose 2/ Dose 3/ Dose 4/
well 1 well 2 well 2 well 2
1 Seed cells to be 30–60%
Adherent cells 2.5 x 104–7.5 x 104
confluent at transfection
2 Prepare reagent mix: Dilute 2 Opti-MEM I
25 µL 25 µL 25 µL 25 µL
volumes of Lipofectamine Stem medium
Reagent in Opti-MEM I Reduced Lipofectamine
Serum Medium 1 µL 1 µL 2 µL 2 µL
Stem reagent
3 Dilute DNA: Dilute 2 volumes of Opti-MEM I
25 µL 25 µL 25 µL 25 µL
DNA in Opti-MEM I Medium medium
DNA 250 ng 500 ng 250 ng 500 ng
4 Add diluted DNA to diluted Reagent mix 25 µL 25 µL 25 µL 25 µL
Lipofectamine Stem Reagent to
each tube in a 1:1 ratio Dilute DNA 25 µL 25 µL 25 µL 25 µL
4a Incubate DNA–reagent complex 10 minutes at room temperature
5 Add DNA–reagent complex DNA–lipid complex 50 µL 50 µL 50 µL 50 µL
to cells Final DNA used 250 ng 500 ng 250 ng 500 ng
Final
Lipofectamine 1 µL 1 µL 2 µL 2 µL
Stem reagent used
6 Incubate transfected cells Incubate and monitor cells for 1–2 days at 37°C
7 Visualize/analyze The following day, overlay an additional 0.5 mL StemPro
transfected cells NSC SFM per well, if NSCs are going to be transfected
for 48 hours
* Visit thermofisher.com/lipofectaminestem for additional information on setting up mRNA or ribonucleoprotein (RNP) transfections in NSCs with Lipofectamine Stem reagent.

112
 Transfection

Expected results
A B

Figure 25-3. Plasmid DNA delivered by Lipofectamine Stem reagent

in NSCs. (A) GFP expression demonstrating 60% transfection efficiency
and (B) brightfield image. NSCs are shown 24 hours posttransfection
with 500 ng of a 6 kb EF1α-GFP plasmid and 1 µL of Lipofectamine Stem
reagent in StemPro NSC SFM on Gibco™ Geltrex™ matrix.

113
Transfection

26 Transfecting neural cells using the

Neon Transfection System

Summary Required materials

The Invitrogen™ Neon™ Transfection System is a benchtop Cells

electroporation device that uses a pipette tip as an • Neural cell line of interest
electroporation chamber to efficiently transfect mammalian
cells including primary cells and stem cells. Media and reagents
• Growth medium and growth factors appropriate for your
Instructions for using the Neon Transfection System for neural cell line
transfecting neural cells are described below. For detailed
• Plasmid DNA of interest (1–5 μg/mL in deionized water
instructions, including several cell-specific protocols, refer
or TE buffer)
to thermofisher.com/neon
• Gibco™ DPBS, no calcium, no magnesium
For detailed information on culture conditions for various (Cat. No. 14190144)
neural cell lines, refer to the instructions supplied with the
specific cell line you are using. • Invitrogen Neon™ Transfection System
(Cat. No. MPK5000)
View this protocol online and order products at
• Invitrogen™ Neon™ Transfection System 10 μL Kit
thermofisher.com/neuroprotocol/neon
(Cat. No. MPK1096) or Neon™ Transfection System
100 μL Kit (Cat. No. MPK10096)

• Appropriate tissue culture plates and supplies

• Gibco™ StemPro™ NSC SFM (Cat. No. A1050901: This

kit contains KnockOut™ DMEM/F-12 Basal Medium
stored at 4°C; StemPro™ NSC SFM Supplement stored
at –20 to –5°C in the dark; and bFGF Recombinant
Human and EGF Recombinant Human proteins stored
at 4°C, desiccated.)

• Gibco™ Astrocyte Medium (Cat. No. A1261301: This

kit contains N-2 Supplement (100X) stored at –20°C;
Dulbecco’s Modified Eagle Medium (DMEM) (1X) stored
at 2°C to 8°C; and One Shot™ Fetal Bovine Serum,
Certified, stored at –20°C in the dark.)

114
 Transfection

Preparing media Transfection protocol

Use complete StemPro NSC SFM for human neural stem Use this procedure to transfect plasmid DNA into human
cells or rat fetal neural stem cells and complete astrocyte NSCs in a 24-well format using the 10-μL Neon kit. All
medium for human astrocytes, rat primary cortical amounts and volumes are given on a per-well basis.
astrocytes, or rat glial precursor cells. Also see “Culture
Conditions” on page 103 of the previous chapter for 1. Cultivate the required number of cells (see table on
additional information. the next page) in the appropriate growth medium
such that the cells are 70–90% confluent on the day
Complete StemPro NSC SFM of the experiment.
To prepare 100 mL of complete StemPro NSC SFM,
aseptically mix the components listed in the table below. 2. On the day of the experiment, harvest and wash cells in
Complete medium is stable for up to 4 weeks when stored DPBS, no calcium, no magnesium (DPBS –/–).
in the dark at 4°C.
3. Resuspend the cell pellet in Resuspension Buffer R
Component Final conc. Amount (included with Neon kits) at the appropriate final density
KnockOut DMEM/F-12 1X 97 mL (see the table on the next page).
GlutaMAX Supplement 2 mM 1 mL
bFGF 20 ng/mL 2 μg 4. Prepare 24-well plates by filling the wells with 0.5 mL
EGF 20 ng/mL 2 μg of the appropriate growth medium without antibiotics
StemPro NSC SFM 2% 2 mL and pre-incubate plates at 37°C in a humidified
Supplement 5% CO2 incubator. If using other plate formats, adjust
the volume accordingly.
Complete astrocyte medium
Use for human astrocytes, rat primary cortical 5. Turn on the Neon unit and enter the following
astrocytes, or rat glial precursor cells electroporation parameters in the Input window.
To prepare 100 mL of complete astrocyte medium, Alternatively, press the Database button and select the
aseptically mix the components listed in the table below. appropriate transfection protocol (if you have already
Complete medium is stable for up to 2 weeks when stored added the electroporation parameters for your cell type).
in the dark at 4°C. For detailed instructions, refer to the manual supplied
with the Neon unit.
Component Final conc. Amount
DMEM 1X 89 mL
N-2 Supplement 1X 1 mL
FBS 10% 10 mL
EGF 20 ng/mL 2 μg
Note: Adding EGF at a final concentration of 20 ng/mL can increase proliferation, but may result in
morphological and phenotypic changes in human astrocytes.

115
Transfection

Pulse voltage Pulse width Neon

Cell type Cell density Pulse number
(V) (ms) Tip
1,400 20 2
Human neural stem cells 1 x 107 cells/mL 1,600 20 1 10 μL
1,700 20 1
1,100 30 1
Human astrocytes 1 x 107 cells/mL 10 μL
1,200 40 1
1,300 20 2
Rat fetal neural stem cells 1 x 107 cells/mL 1,500 10 3 10 μL
1,600 10 3
1,400 20 2
Rat primary cortical astrocytes 0.5 x 107 cells/mL 1,400 30 1 10 μL
1,700 20 1
1,300 10 3
Rat glial precursor cells 1 x 107 cells/mL 10 μL
1,500 20 1

1. Fill the Neon Tube with 3 mL of Buffer E. (Use Buffer E2 if 6. Press the push-button on the Neon Pipette to the first
you are using the 100 μL Neon Tip.) stop and immerse the Neon Tip into the cell–DNA
mixture. Slowly release the push-button on the pipette to
2. Insert the Neon Tube into the Neon Pipette Station aspirate the cell–DNA mixture into the Neon Tip.
until you hear a click, indicating that the tube has
locked in position. 7. Insert the Neon Pipette with the sample vertically into
the Neon Tube placed in the Neon Pipette Station
3. Transfer 0.5 μg of plasmid DNA into a sterile, 1.5 mL until you hear a click, indicating that the pipette has
microcentrifuge tube. locked in position.

Note: The quality and concentration of DNA used 8. Ensure that you have entered the appropriate
for electroporation plays an important role for the electroporation parameters and press Start on the
transfection efficiency. We strongly recommend using Neon touchscreen.
high-quality plasmid purification kits such as Invitrogen™
PureLink™ HiPure plasmid DNA purification kits to The Neon device delivers the electric pulse
prepare DNA. according to the parameters entered in step 5, and
the touchscreen displays Complete to indicate that
4. Add 1 mL of cells (resuspended in step 3) to the tube electroporation is complete.
containing the plasmid DNA and gently mix.
9. Remove the Neon Pipette from the Neon Pipette
5. Insert a 10 μL Neon Tip into the Neon Pipette. Station and immediately transfer the samples
from the Neon Tip into the prepared culture plate
containing the appropriate pre-warmed complete
growth medium without antibiotics.

116
 Transfection

10. Discard the Neon Tip into an appropriate biological Expected results
hazardous waste container.
Human Neural Stem Cells
11. Repeat steps 5–10 for the remaining samples. Gibco™ Human Neural Stem Cells (H9-Derived)
(Cat. No. N7800100), cultured in complete StemPro NSC
12. Gently rock the plate to assure even distribution of SFM, were transfected with 0.5 μg of a plasmid encoding
the cells. Incubate the plate at 37°C in a humidified the Emerald Green Fluorescent Protein (EmGFP) using the
5% CO2 incubator. Neon Transfection System with the parameters listed in the
following table. 48 hours posttransfection, the cells were
13. Assay the samples to determine the transfection analyzed by light (A) and fluorescence microscopy (B).
efficiency (e.g., fluorescence microscopy
A B
or functional assay).

Cell
Pulse Pulse
density Pulse Transfection Neon
voltage width Viability
(cells/ number efficiency Tip
(V) (ms)
mL)
1,400 20 2 82% 95%
1 x 107 1,600 20 1 84% 95% 10 μL
1,700 20 1 87% 96%

117
Transfection

Human Astrocytes Rat Fetal Neural Stem Cells

Gibco™ Human Astrocytes (Cat. No. N7805100) were Gibco™ Rat Fetal Neural Stem Cells (Cat. No. N7744100)
transfected using the Neon Transfection System were transfected using the Neon Transfection System
and 0.5 μg of a plasmid encoding the Emerald and 0.5 μg of a plasmid encoding the Emerald
Green Fluorescent Protein (EmGFP); 24 hours Green Fluorescent Protein (EmGFP); 24 hours
post-electroporation, the cells were analyzed by light (A) post-electroporation, the cells were analyzed by light (A)
and fluorescence microscopy (B). and fluorescence microscopy (B).

A B A B

Cell Cell
Pulse Pulse Pulse Pulse
density Pulse Transfection Neon density Pulse Transfection Neon
voltage width Viability voltage width Viability
(cells/ number efficiency Tip (cells/ number efficiency Tip
(V) (ms) (V) (ms)
mL) mL)
1,100 30 1 92% 97% 1,100 30 1 92% 97%
1 x 107 10 μL 1 x 107 10 μL
1,200 40 1 93% 97% 1,200 40 1 93% 97%

118
 Transfection

Rat Primary Cortical Astrocytes Rat Glial Precursor Cells

Gibco™ Rat Primary Cortical Astrocytes (Cat. No. N7745100) Gibco™ Rat Glial Precursor Cells (Cat. No. N7746100)
were transfected using the Neon Transfection System were transfected using the Neon Transfection System
and 0.5 μg of a plasmid encoding the Emerald and 0.5 μg of a plasmid encoding the Emerald
Green Fluorescent Protein (EmGFP); 24 hours Green Fluorescent Protein (EmGFP); 24 hours
post-electroporation, the cells were analyzed by light (A) post-electroporation, the cells were analyzed by light (A)
and fluorescence microscopy (B). and fluorescence microscopy (B).

A B A B

Cell Cell
Pulse Pulse Pulse Pulse
density Pulse Transfection Neon density Pulse Transfection Neon
voltage width Viability voltage width Viability
(cells/ number efficiency Tip (cells/ number efficiency tip
(V) (ms) (V) (ms)
mL) mL)
1,400 20 2 69% 87% 1,300 10 3 49% 78%
1 x 107 10 μL
0.5 x 107 1,400 30 1 71% 89% 10 μL 1,500 20 1 44% 64%
1,700 20 1 71% 90%

119
Transfection

Troubleshooting

For troubleshooting tips regarding the Neon Transfection

System, see the table below. For troubleshooting tips
regarding the culture and passaging of your cells, refer to
the manual provided with the cells.

Problem Possible cause Solution

• Make sure that the Neon Tip is inserted into Neon
No Neon Tip is inserted, or the Neon
Connection failure Pipette correctly as described. There should be no gap
Tip is inserted incorrectly
between the tip and the top head of the pipette.
• Avoid any air bubbles in the Neon Tip while
Air bubbles in the Neon Tip
Arcing (sparks) aspirating the sample.
High voltage or pulse length settings • Reduce the voltage or pulse length settings.
• Use high-quality plasmid DNA for transfection (use
high-quality plasmid purification kits such as PureLink
HiPure plasmid DNA purification kits) to prepare DNA.
• Resuspend the purified DNA in deionized water or
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at a
concentration between 0.5–5 μg/μL.
Poor DNA quality • Check the purity of the purified DNA preparation by
measurement of the A 260/A 280 ratio. The ratio should be
at least 1.8 for electroporation.
• Do not precipitate DNA with ethanol to concentrate
DNA. DNA concentrated by ethanol precipitation shows
poor transfection efficiency and cell viability due to
Low cell survival rate salt contamination.
• Avoid severe conditions during cell harvesting,
especially high-speed centrifugation, and
pipet cells gently.
• Avoid using over-confluent cells or cells at high
Cells are stressed or damaged
densities, as this may affect the cell survival
after electroporation.
• After electroporation, immediately plate the cells into
pre-warmed culture medium without antibiotics.
• Do not use the same Neon Tip for electroporation
more than 2 times, because the repeated application
Multiple use of the same Neon Tip
of electric pulses reduces the tip quality and impairs
its physical integrity.

120
 Transfection

Problem Possible cause Solution

Poor plasmid DNA quality or the • Use high-quality plasmid DNA for transfection.
plasmid DNA amount is low • Start with 0.5 μg plasmid DNA per sample.
• Use the recommended cell densities of 1 x 105 cells per
Incorrect cell density
10 μL per sample (i.e., 1 x 107 cells/mL).
Low transfection • Use the recommended voltage, pulse width, and pulse
efficiency number. We recommend optimizing the electroporation
Incorrect electroporation parameters
parameters using the preprogrammed 24-well
optimization protocol available on the Neon unit.
• Test cells for Mycoplasma contamination. Start a new
Mycoplasma-contaminated cells
culture from a fresh stock.
Inconsistent cell confluency or • Always use cells with low passage number and harvest
passage number cells with comparable confluency levels.
• Do not use the same Neon Tip for more than 2 times
because the repeated application of electric pulses
Nonreproducible reduces the tip quality and impairs its physical integrity.
transfection
efficiency Multiple use of the same Neon Tip or • Do not use the same Neon Tube for more than
the same Neon Tube 10 times.
• Always use a new Neon Tip and Neon Tube
for different plasmid DNA samples to avoid
any cross-contamination.

121
122
Appendix
A1 Thermo Fisher Scientific products

Overview

Thermo Fisher Scientific offers products that are designed cell culture media products available from Thermo Fisher
to meet your neural cell culture needs, including Gibco™ Scientific are tested for contamination to help ensure their
cell culture media, reagents, sera, and growth factors. All quality, safety, consistency, and regulatory compliance.

Ordering information

Cells

Product Quantity Cat. No.

Human

Human Astrocytes 1 mL N7805100

Human Episomal iPSC line 1 x 106 cells⁄vial A18945

StemPro Neural Stem Cells 1 x 106 cells A15654

Mouse

1 x 106 cells A15585

Primary Mouse Cortical Neurons
4 x 106 cells A15586

Primary Mouse Hippocampal Neurons 1 x 106 cells A15587

Rat

1 x 106 cells A36511

Primary Rat Cortex Neurons, Sprague-Dawley
4 x 106 cells A36512

1 x 106 cells A1084001

Primary Rat Cortical Neurons
4 x 106 cells A1084002

Primary Rat Hippocampal Neurons 1 mL A1084101

Rat

Rat Fetal Neural Stem Cells 1 mL N7744100

Rat Glial Precursor Cells 1 mL N7746100

123
Appendix

Cells

Product Quantity Cat. No.

Rat Primary Cortical Astrocytes 1 x 106 cells N7745100

Media and supplements

Product Quantity Cat. No.

500 mL 12634010
Advanced DMEM/F-12
10 x 500 mL 12634028

Astrocyte Medium 500 mL A1261301

B-27 Plus Neuronal Culture System 1 system A3653401

B-27 Plus Supplement (50X) 10 mL A3582801

10 mL 17504044
B-27 Supplement (50X), serum free
100 mL 17504001

CTS B-27 Supplement, XenoFree 10 mL A1486701

CTS DPBS, calcium, magnesium 1,000 mL A1285801

CTS (Cell Therapy Systems) DPBS, without calcium chloride, without
1L A1285601
magnesium chloride
CTS GlutaMAX-I Supplement 100 mL A1286001

CTS KnockOut DMEM/F-12 500 mL A1370801

CTS N-2 Supplement 5 mL A1370701

CultureOne Supplement (100X) 5 mL A3320201

Dulbecco’s Modified Eagle Medium (DMEM), high glucose, pyruvate* 500 mL 11995065

500 mL 10565018
DMEM/F-12, GlutaMAX supplement
10 x 500 mL 10565042

Essential 8 Medium 500 mL A1517001

Essential 8 Flex Medium Kit 1 kit A2858501

100 mL 16141061
Fetal Bovine Serum (FBS), embryonic stem cell–qualified, US origin*
500 mL 16141079

100 mL 35050061
GlutaMAX Supplement
20 x 100 mL 35050079

124
 Appendix

Media and supplements

Product Quantity Cat. No.

100 mL 16210064
Goat Serum, New Zealand origin*
500 mL 16210072

Hibernate-E Medium 500 mL A1247601

5 mL 17502048
N-2 Supplement (100X)
50 mL 17502001

Neurobasal Medium 500 mL 21103049

Neurobasal Plus Medium 500 mL A3582901

100 mL 31985062

Opti-MEM I Reduced Serum Medium 500 mL 31985070

10 x 500 mL 31985088

PSC Dopaminergic Neuron Differentiation Kit 1 kit A3147701

PSC Neural Induction Medium 500 mL A1647801

RevitaCell Supplement (100X) 5 mL A2644501

StemPro NSC SFM 1 kit A1050901

Substrates, matrices, and bioscaffolds

Product Quantity Cat. No.

CELLstart CTS Substrate 2 mL A1014201

Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement 1 mL A1413301

Membrane Matrix 5 mL A1413302

1 mL A1413201
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix
5 mL A1413202

Laminin Mouse Protein, Natural 1 mg 23017015

Poly-D-Lysine 100 mL A3890401

1 mL A14700
Vitronectin (VTN-N) Recombinant Human Protein, Truncated
10 mL A31804

125
Appendix

Reagents

Product Quantity Cat. No.

2-Mercaptoethanol (55 mM) (β-Mercaptoethanol) 50 mL 21985023

20 mL 15240096

Antibiotic-Antimycotic (100X) 100 mL 15240062

20 x 100 mL 15240112

CTS Synth-a-Freeze Medium 50 mL A1371301

CTS TrypLE Select Enzyme 100 mL A1285901

DAPI (4´,6-Diamidino-2-Phenylindole, Dihydrochloride) 10 mg D1306

DMSO, Anhydrous (dimethylsulfoxide) 10 x 3 mL D12345

Glucose Solution 50 mL A2494001

FluoVolt Membrane Potential Kit 1 kit F10488

0.1 mL L3000001

0.75 mL L3000008

Lipofectamine 3000 Transfection Reagent 1.5 mL L3000015

5 x 1.5 mL L3000075

15 mL L3000150

0.3 mL 11668030

0.75 mL 11668027
Lipofectamine 2000 Transfection Reagent
1.5 mL 11668019

15 mL 11668500

0.1 mL STEM00001

0.3 mL STEM00003
Lipofectamine Stem Transfection Reagent
0.75 mL STEM00008

1.5 mL STEM00015

126
 Appendix

Reagents

Product Quantity Cat. No.

0.1 mL LMRNA001

0.3 mL LMRNA003

Lipofectamine MessengerMAX Transfection Reagent 0.75 mL LMRNA008

1.5 mL LMRNA015

15 mL LMRNA150

0.1 mL 13778100

0.3 mL 13778030

Lipofectamine RNAiMAX Transfection Reagent 0.75 mL 13778075

1.5 mL 13778150

15 mL 13778500

Live Cell Imaging Solution (LCIS) 500 mL A14291DJ

NucBlue Fixed Cell ReadyProbes Reagent 1 kit R37606

Penicillin-Streptomycin (5,000 U/mL) 100 mL 15070063

Probenecid, Water Soluble (Cat. No. P36400) 10 x 77 mg P36400

2 mL P10144

ProLong Gold Antifade Mountant 10 mL P36930

5 x 2 mL P36934

PSC Cryopreservation Kit 50 mL A2644601

StemPro Accutase Cell Dissociation Reagent 100 mL A1110501

Trypan Blue Solution, 0.4% 100 mL 15250061

100 mL 12604013

500 mL 12604021
TrypLE Express Enzyme (1X), no phenol red
20 x 100 mL 12604039

5L 12604054

100 mL 12563011
TrypLE Select Enzyme (1X), no phenol red
500 mL 12563029

127
Appendix

Reagents

Product Quantity Cat. No.

100 mL 25300054

Trypsin-EDTA (0.05%), phenol red 500 mL 25300062

20 x 100 mL 25300120

Valinomycin 25 mg V1644

Growth factors, purified proteins, and antibodies**

Product Quantity Cat. No.

5 µg 10908010
BDNF Recombinant Human Protein
10 µg PHC7074

CNTF Recombinant Human Protein 20 µg PHC7015

5 µg 10605HNAE5

10 µg PHG0314

25 µg PHG0315

5 x 5 µg 10605HNAE25
EGF Recombinant Human Protein
50 µg 10605HNAE50

100 µg PHG0311

5 x 50 µg 10605HNAE250

1 mg PHG0313

100 µg CTP0261
FGF-Basic Full Length CTS Recombinant Human Protein
1 mg CTP0263

Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate 500 µL A-11029

Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate 500 µL A-11037

MAP2 Antibody (M13) 100 µg 13-1500

PDGF-AA Recombinant Human Protein 10 µg PHG0035

128
 Appendix

Buffers and balanced salt solutions

Product Quantity Cat. No.

CTS (Cell Therapy Systems) DPBS, calcium, magnesium

1,000 mL A1285801
(CTS Dulbecco’s Phosphate-Buffered Saline (DPBS))
CTS DPBS, without calcium chloride, without magnesium chloride
1L A1285601
(CTS Dulbecco’s Phosphate-Buffered Saline without Ca2+ or Mg2+)
100 mL 14040141

500 mL 14040133
DPBS, calcium, magnesium
1,000 mL 14040117
(Dulbecco’s Phosphate-Buffered Saline with Ca2+ and Mg2+, DPBS +/+)
10 x 500 mL 14040182

6 x 1,000 mL 14040216

500 mL 14190144

1,000 mL 14190136

5L 14190342
DPBS, no calcium, no magnesium
10 x 500 mL 14190250
(Dulbecco’s Phosphate-Buffered Saline without Ca2+ or Mg2+ , DPBS –/–)
6 x 1,000 mL 14190235

10 L 14190359

20 L 14190367

500 mL 14025092

HBSS, calcium, magnesium, no phenol red* 1,000 mL 14025076

(Hanks’ Balanced Salt Solution with Ca2+ and Mg2+ and without phenol red) 10 x 500 mL 14025134

6 x 1,000 mL 14025126

100 mL 14170120
HBSS, no calcium, no magnesium*
500 mL 14170112
(Hanks’ Balanced Salt Solution without Ca2+ or Mg2+)
10 x 500 mL 14170161

Assay kits

Product Quantity Cat. No.

Fluo-4 Calcium Imaging Kit 1 kit F10489

Human Dopaminergic Neuron Immunocytochemistry Kit 40 reactions A29515

LIVE/DEAD Cell Vitality Assay Kit, C12 Resazurin/SYTOX Green 1 kit L34951

129
Appendix

Assay kits

Product Quantity Cat. No.

LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells 1 kit L3224

mMESSAGE mMACHINE T7 ULTRA Transcription Kit 10 reactions AM1345

25 x 2 reactions MPK1025
Neon Transfection System 10 µL Kit
96 x 2 reactions MPK1096

25 x 2 reactions MPK10025
Neon Transfection System 100 µL Kit
96 x 2 reactions MPK10096

100 reactions 11733038

Platinum SYBR Green qPCR SuperMix-UDG
500 reactions 11733046

10 preps 12183020

PureLink RNA Mini Kit 50 preps 12183018A

250 preps 12183025

S (250
TaqMan Gene Expression Assay (FAM) reactions/250 µL), 4331182
inventoried
TaqMan Gene Expression Master Mix 1 x 5 mL 4369016

Plasticware

Product Quantity Cat. No.

Nalgene General Long-Term Storage Cryogenic Tubes (1.0 mL capacity) Case of 500 5000-1012

Nalgene General Long-Term Storage Cryogenic Tubes (1.2 mL capacity) Case of 500 5000-0012

Nalgene General Long-Term Storage Cryogenic Tubes (1.5 mL capacity) Case of 500 5000-1020

Nunc Cell Scrapers (23 cm handle, for use with 25 to 80 cm2 flasks) Case of 250 179693

Nunc Cell Scrapers (32 cm handle, for use with 75 to 175 cm2 flasks) Case of 250 179707

Nunc Cell-Culture Treated Multidishes, 4 well Case of 120 176740

Nunc Edge 2.0 96-Well Treated Plate, Lid Case of 50 167425

Nunc Lab-Tek II Chamber Slide System, 4 well Pack of 16 154526PK

Nunc MicroWell 96-Well Optical-Bottom Plates with Polymer Base, cell
Case of 30 165305
culture, black
6 units/bag,
Nunclon Sphera Flasks (T-25 Cell Culture Flask) 174951
18 units/case

130
 Appendix

Plasticware

Product Quantity Cat. No.

4 units/bag,
Nunclon Sphera Flasks (T-75 Cell Culture Flask) 174952
24 units/case
1 unit/pack,
Nunclon Sphera Dishes (Multidish 6-Well) 174932
7 units/case
T-25 Nunclon Sphera EasYFlask Case of 18 174951

T-75 Nunclon Sphera EasYFlask Case of 24 174952

Instruments and software

Product Quantity Cat. No.

Real-Time PCR Systems thermofisher.com/qpcr

Attune NxT Flow Cytometer thermofisher.com/attune

CellInsight CX5 High-Content Screening (HCS) Platform thermofisher.com/hcs

Countess II Automated Cell Counter thermofisher.com/countess

EVOS FL Auto Imaging System thermofisher.com/evosflauto

EVOS FL Imaging System thermofisher.com/evosfl

HCS Studio 2.0 Cell Analysis Software, client installation 1 each SX000041A

Neon Transfection System 1 each MPK5000

Accessory products

Product Quantity Cat. No.

BlueJuice Gel Loading Buffer (10X) 3 x 1 mL 10816-015

Custom primers thermofisher.com/oligos

100 mL 15230170

Distilled Water 500 mL 15230162

1,000 mL 15230147

EVOS Light Cube, GFP 1 each AMEP4651

25 covers 4360954
MicroAmp Optical Adhesive Film
100 covers 4311971

Mr. Frosty Freezing Container (1.0 to 2.0 mL tube capacity) 1 each 5100-0001

131
Appendix

Accessory products

Product Quantity Cat. No.

1 x 100 mL AM9938

5 x 100 mL AM9939

10 x 50 mL AM9937
Nuclease-Free Water (not DEPC-Treated)
1 x 500 mL AM9930

1 x 1,000 mL AM9932

4 x 1,000 mL 4387936

30 units 18021014
Ribonuclease H (RNase H)
120 units 18021071

Silencer Select siRNAs thermofisher.com/sirna

50 reactions 18091050
SuperScript IV First-Strand Synthesis System
200 reactions 18091200
* For In Vitro Diagnostic Use.
** For additional antibodies, see pages 74–75 and 79–80.

132
 Appendix

A2 Resources for more information

Books
• Neural Stem Cell Assays, edited by Navjot Kaur and Mohan Vemuri, Wiley Press, 2015.

• Developmental Biology, 9th edition, edited by Scott F. Gilbert Sinauer Associates, 2010.

• Neural Stem Cells, 2nd edition, edited by Leslie P. Weiner, Humana Press, 2008.

• Neural Development and Stem Cells, 2nd edition, edited by Mahendra S. Rao, Humana Press, 2006.

• Protocols for Neural Cell Culture, 4th edition, edited by Laurie C. Doering, Humana Press, 2010.

• Protocols for Neural Cell Culture, 3rd edition, edited by Sergey Fedoroff and Arleen Richardson, Humana Press, 2001.

Journals
• Neuron, cell.com/neuron

• Development, dev.biologists.org

• The Journal of Neuroscience, jneurosci.org

• European Journal of Neuroscience, onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291460-9568

• Developmental Neurobiology, onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291097-4695

• Nature Reviews Neuroscience, nature.com/nrn/index.html

• Cell Stem Cell, cell.com/cell-stem-cell/home

Organizations
• American Academy of Neurology, aan.com

• Society for Neuroscience, sfn.org

• European Neuroscience and Society Network, lse.ac.uk/collections/ENSN

• Federation of European Neuroscience Societies, fens.mdc-berlin.de

• Japanese Neuroscience Society, jnss.org/en/

• International Society for Stem Cell Research, isscr.org

133
Appendix

Government sites
• National Institute of Neurological Disorders and Stroke (NINDS), ninds.nih.gov

• National Institute of Mental Health (NIMH), nimh.nih.gov

• Food and Drug Administration (FDA), fda.gov

• National Institute of Child Health and Human Development (NICHD), nichd.nih.gov

• NCBI PubMed, ncbi.nlm.nih.gov/pubmed

• National Institutes of Health Entrez Databases, ncbi.nlm.nih.gov/Database/index.html

• National Library of Medicine’s MEDLINEplus, ncbi.nlm.nih.gov/Database/index.html

Websites
• The Dana Foundation, dana.org

• Neuromuscular Disease Center at Washington University School of Medicine, St. Louis, neuromuscular.wustl.edu

• Neuroscience Information Framework, neuinfo.org

134
 Appendix

A3 Technical support

Web resources SDS

Visit the thermofisher.com website for: SDSs are available at

thermofisher.com/sds
• Technical resources, including manuals, vector maps
and sequences, application notes, Safety Data Sheets
(SDSs), FAQs, formulations, citations, and handbooks. Certificate of Analysis
• Complete technical support contact information
The Certificate of Analysis (CoA) provides detailed
• Access to the Thermo Fisher Scientific online catalog quality control and product qualification information for
each product. CoAs are available on our website. Go
• Additional product information and special offers
to thermofisher.com/support and search for the CoA
• Warranty information by product lot number, which is printed on the product
packaging (tube, pouch, or box).

Contact us

For more information or technical assistance, please

consult our website at thermofisher.com/contactus

135
Find out more at thermofisher.com/neuroprotocols
For Research Use Only. Not for use in diagnostic procedures. © 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries
unless otherwise specified. Pluronic is a trademark of BASF Corporation. Parafilm is a trademark of Bemis Company, Inc. Corning and BioCoat are trademarks of Corning, Inc. Essential 8 is a trademark
of Cellular Dynamics International. MetaFluor is a trademark of Molecular Devices, LLC. Nikon is a trademark of Nikon Corp. Hybri-Max is a trademark of Sigma-Aldrich Co. LLC. B-27 is a trademark of
Southern Illinois University. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Triton is a trademark of Union Carbide Chemicals and Plastics Technology
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