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Metastasis Research Protocols 2nd ed. 2014 Edition
Edition Miriam Dwek Digital Instant Download
Author(s): Miriam Dwek, Udo Schumacher, Susan A. Brooks
ISBN(s): 9781461482437, 1461482437
Edition: 2nd ed. 2014 Edition
File Details: PDF, 4.42 MB
Year: 2013
Language: english
Methods in
Molecular Biology 1070

Miriam Dwek
Udo Schumacher
Susan A. Brooks Editors

Metastasis
Research
Protocols
Second Edition
METHODS IN M O L E C U L A R B I O LO G Y ™

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

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Metastasis Research Protocols

Second Edition

Edited by

Miriam Dwek
Molecular and Applied Biosciences, School of Life Sciences, University of Westminster, London, UK

Udo Schumacher
Institute Anatomie und Experimentelle Morphologie, Universitätsklinikum Hamburg-Eppendorf,
Hamburg, Germany

Susan A. Brooks
Department of Biological and Medical Sciences, Oxford Brookes University, Headington, Oxford, UK
Editors
Miriam Dwek Udo Schumacher
Molecular and Applied Biosciences Institute Anatomie und Experimentelle
School of Life Sciences Morphologie
University of Westminster Universitätsklinikum Hamburg-Eppendorf
London, UK Hamburg, Germany

Susan A. Brooks
Department of Biological and Medical Sciences
Oxford Brookes University
Headington, Oxford, UK

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-4614-8243-7 ISBN 978-1-4614-8244-4 (eBook)
DOI 10.1007/978-1-4614-8244-4
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2013947409

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Preface

Diverse molecular, cellular, and environmental events must all come together to allow the suc-
cessful formation of secondary cancers, metastases. The vast amount of knowledge that has
been amassed in relation to the biology underlying cancer formation is being applied to enable
a better understanding of the metastatic process. It is well accepted that elucidation of the key
events in the process will lead to the next generation of laboratory tests for early diagnosis of
metastases and for the treatment of occult as well as more clinically advanced disease.
This second edition of Metastasis Research Protocols brings together updated versions of
the seminal techniques that were presented in the first edition and also includes new tech-
niques that have recently been shown to be important in illuminating the processes under-
lying this important area of biology.
The first volume of Metastasis Research Protocols, Analysis of Cells and Tissues, takes the
reader through key cellular and molecular techniques relevant to exploration of cancer cells
and tissues. In this second volume, Models of Metastasis, we move to the level of living cells and
tissues, and present methodologies applicable to behaviour in vitro, in animal models and in
mathematical constructs. It is concerned with the interaction between cancer cells and their
host/environment. The focus throughout is on the tools that have been shown to be help-
ful in unravelling the processes important in cancer metastasis.
In comparison to the first edition of Metastasis Research Protocols, here we have retained
important key methods in both volumes and introduced new and cutting-edge methods
which are making an impact in the arena of metastasis research. Consistent with the first
edition, volume 1 includes standard techniques including immunochemistry (Chapters 1,
2, 3, and 4), PCR (Chapter 5), and SDS-PAGE (Chapter 6), the mainstay of many labora-
tories, and these have been revised and updated. Volume 1 has also been extended to incor-
porate newer techniques, for example, affinity measurement of biomolecular interactions
(Chapter 11), methylation analysis of microRNA (Chapter 15), and RNAi technology
(Chapter 16). This volume, volume 2, similarly retains chapters from the first edition,
where they represent methods commonly and usefully employed in metastasis research, and
these have been fully revised and updated. They include, for example, methods to assess
cancer cell adhesion to extracellular matrix components and endothelial cells (Chapters 1,
2, 3, 4, and 5), and syngeneic and xenograft animal models of metastasis (Chapters 9, 10,
16 and 17). In addition, new methods are presented, for example, for the production of in
vivo double knockout models (Chapter 14) and the application of fluorescent imaging
techniques for monitoring the development of metastases in vivo (Chapters 11 and 12).
Each chapter stands alone and we aim for there to be enough detail for them to be useful
to the newcomer and the experienced researcher alike.
One of the much-valued aspects of the Methods in Molecular Biology series is that it aims
to impart knowledge of complex methodology to the end-user in an accessible manner. The
“Notes” section found at the end of each chapter serves to demystify the techniques in a

v
vi Preface

handy “hints and tips” format, which enables researchers who may be hesitant to adopt a
new procedure to try it out, thereby adding to their repertoire of laboratory techniques. We
have tried to maintain this key element in the chapters presented in these two volumes and
we hope that you find this to be a continued useful aspect of the series.
Finally, we would like to thank all of our contributors who have worked tirelessly to
master their techniques, for sharing these with us and you, the reader. We hope that you
find in these two volumes methods that will assist in helping you to make new observations
that will enhance our knowledge and understanding of the complexities of metastasis and
may, in turn, lead to developments in treatments aimed at combating cancer metastasis.

London, UK Miriam Dwek

Hamburg, Germany Udo Schumacher
Oxford, UK Susan A. Brooks
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 In Vitro Invasion Assay Using Matrigel™:

A Reconstituted Basement Membrane Preparation . . . . . . . . . . . . . . . . . . . . . 1
Debbie M.S. Hall and Susan A. Brooks
2 Single Cell and Spheroid Collagen Type I Invasion Assay . . . . . . . . . . . . . . . . 13
Olivier De Wever, An Hendrix, Astrid De Boeck, Frank Eertmans,
Wendy Westbroek, Geert Braems, and Marc E. Bracke
3 Rocking Adhesion Assay System to Study Adhesion
and Transendothelial Migration of Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . 37
Deepashree Bapu, Munira Khadim, and Susan A. Brooks
4 Small-Cell Lung Cancer (SCLC) Cell Adhesion on E- and P-Selectin
Under Physiological Flow Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Ulrich Richter
5 Adhesion of Tumor Cells to Matrices and Endothelium. . . . . . . . . . . . . . . . . . 57
Clara M. Yates, Helen M. McGettrick, Gerard B. Nash, and G.Ed Rainger
6 Cell Aggregation Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Delphine Debruyne, Tom Boterberg, and Marc E. Bracke
7 Chick Heart Invasion Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Marc E. Bracke, Virinder S. Parmar, Anthony L. DePass,
Christian V. Stevens, Barbara W. Vanhoecke, and Marc M. Mareel
8 Computer Simulation of the Metastatic Progression . . . . . . . . . . . . . . . . . . . . 107
Gero Wedemann, Anja Bethge, Volker Haustein, and Udo Schumacher
9 Theoretical Considerations in Using Animal Models of Metastasis
and Brief Methodology for In Vivo Colorectal Cancer Models
in SCID and Nude Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Sue A. Watson and Rajendra Kumari
10 Syngeneic Murine Metastasis Models: B16 Melanoma . . . . . . . . . . . . . . . . . . . 131
Raffaella Giavazzi and Alessandra Decio
11 Imageable Clinically Relevant Mouse Models of Metastasis . . . . . . . . . . . . . . . 141
Robert M. Hoffman
12 Imaging Metastatic Cell Trafficking at the Cellular Level
In Vivo with Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Robert M. Hoffman

vii
viii Contents

13 Ultrasound Techniques for the Detection of Tumors and Metastases

in Small Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Michael Didié and Wolfram-Hubertus Zimmermann
14 The PFP/RAG2 Double-Knockout Mouse in Metastasis
Research: Small-Cell Lung Cancer and Prostate Cancer. . . . . . . . . . . . . . . . . . 191
Imke Müller and Sebastian Ullrich
15 Ultrasound-Guided Intracardial Injection and In Vivo Magnetic
Resonance Imaging of Single Cells in Mice as a Paradigm
for Hematogenous Metastases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Johannes Salamon and Kersten Peldschus
16 Magnetic Resonance Imaging of Metastases in Xenograft
Mouse Models of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Kersten Peldschus and Harald Ittrich
17 Spontaneous and Experimental Metastasis Models: Nude Mice . . . . . . . . . . . . 223
Janet E. Price
18 Identifying the Origin and Phenotype of Cells in Tumor Xenografts . . . . . . . . 235
Rosemary Jeffery, Pooja Seedhar, and Richard Poulsom

Erratum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E1
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Contributors

GERHARD ADAM • Department of Diagnostic and Interventional Radiology, University

Medical Center Hamburg-Eppendorf, Hamburg, Germany
DEEPASHREE BAPU • Department of Biological and Medical Sciences, Oxford Brookes
University, Headington, Oxford, UK
ANJA BETHGE • University of Applied Sciences Stralsund, Stralsund, Germany
ASTRID DE BOECK • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium
TOM BOTERBERG • Laboratory of Experimental Cancer Research, Department of
Radiotherapy and Nuclear Medicine, Ghent University Hospital, Ghent, Belgium
MARC E. BRACKE • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium
GEERT BRAEMS • Department of Gynaecology, Ghent University Hospital, Ghent, Belgium
SUSAN A. BROOKS • Department of Biological and Medical Sciences, Oxford Brookes University,
Headington, Oxford, UK
DELPHINE DEBRUYNE • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium
ALESSANDRA DECIO • Department of Oncology, Laboratory of Biology and Therapy
of Metastasis, Mario Negri Institute for Pharmacological Research, Bergamo, Italy
ANTHONY L. DEPASS • Department of Biology, Long Island University-Brooklyn,
Brooklyn, NY, USA
MICHAEL DIDIÉ • Department of Pharmacology, Georg-August-University,
Goettingen, Germany
FRANK EERTMANS • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium
RAFFAELLA GIAVAZZI • Department of Oncology, Laboratory of Biology and Therapy
of Metastasis, Mario Negri Institute for Pharmacological Research, Bergamo, Italy
DEBBIE M.S. HALL • Department of Biological and Medical Sciences, Oxford Brookes
University, Headington, Oxford, UK
VOLKER HAUSTEIN • System Engineering and Information Management, Institute for
Applied Computer Science, University of Applied Sciences Stralsund, Stralsund, Germany
AN HENDRIX • Department of Medical Oncology, Ghent University Hospital, Ghent, Belgium
ROBERT M. HOFFMAN • AntiCancer, Inc., San Diego, CA, USA
HARALD ITTRICH • Department of Diagnostic and Interventional Radiology, University
Medical Center Hamburg-Eppendorf, Hamburg, Germany
ROSEMARY JEFFERY • Molecular Pathology Facility, National Centre for Bowel Research
and Surgical Innovation, Centre for Digestive Diseases, Blizard Institute Barts
and The London School of Medicine and Dentistry, Queen Mary University of London,
London, UK
MUNIRA KHADIM • Department of Biological and Medical Sciences, Oxford Brookes University,
Headington, Oxford, UK

ix
x Contributors

RAJENDRA KUMARI • School of Clinical Sciences, Queen’s Medical Centre, Nottingham, UK

CLAUDIA LANGE • Department of Bone Marrow Transplantation, University Medical
Center Hamburg-Eppendorf, Hamburg, Germany
ULRICH LEHMANN • Institute of Pathology, Medizinische Hochschule Hannover,
Hannover, Germany
MARC M. MAREEL • Department of Radiotherapy, Laboratory of Experimental
Cancer Research, Ghent University Hospital, Ghent, Belgium
HELEN M. MCGETTRICK • School of Clinical and Experimental Medicine, College of
Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK
IMKE MÜLLER • Institute for Anatomy, Experimental Morphology, University Medical
Center Hamburg-Eppendorf, Hamburg, Germany
GERARD B. NASH • School of Clinical and Experimental Medicine, College of Medical and
Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK
VIRINDER S. PARMAR • Department of Chemistry, Bioorganic Laboratory, University of
Delhi, Delhi, India
KERSTEN PELDSCHUS • Department of Diagnostic and Interventional Radiology, University
Medical Center Hamburg-Eppendorf, Hamburg, Germany
RICHARD POULSOM • Molecular Pathology Facility, National Centre for Bowel Research
and Surgical Innovation, Centre for Digestive Diseases, Blizard Institute, Barts
and The London School of Medicine and Dentistry, Queen Mary University of London,
London, UK
JANET E. PRICE • Department of Cancer Biology, M.D., Anderson Cancer Centre, Houston,
TX, USA
G. ED RAINGER • School of Clinical and Experimental Medicine, College of Medical and
Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK
ULRICH RICHTER • Institute for Anatomy II: Experimental Morphology, University Medical
Center Hamburg-Eppendorf, Hamburg, Germany
JOHANNES SALAMON • Department of Diagnostic and Interventional Radiology, University
Medical Center Hamburg-Eppendorf, Hamburg, Germany
UDO SCHUMACHER • Institute Anatomie und Experimentelle Morphologie,
Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany
POOJA SEEDHAR • Molecular Pathology Facility, National Centre for Bowel Research
and Surgical Innovation, Centre for Digestive Diseases, Blizard Institute, Barts
and The London School of Medicine and Dentistry, Queen Mary University of London,
London, UK
CHRISTIAN V. STEVENS • Department of Organic Chemistry, Faculty of Bioscience
Engineering, Ghent University, Ghent, Belgium
SEBASTIAN ULLRICH • Institute for Anatomy, Experimental Morphology, University Medical
Center Hamburg-Eppendorf, Hamburg, Germany
BARBARA W. VANHOECKE • Department of Radiotherapy, Laboratory of Experimental
Cancer Research, Ghent University Hospital, Ghent, Belgium
SUE A. WATSON • School of Clinical Sciences, Queen’s Medical Centre, Nottingham, UK
GERO WEDEMANN • University of Applied Sciences Stralsund, Stralsund, Germany
WENDY WESTBROEK • National Human Genome Research Institute, NIH, Bethesda,
MD, USA
OLIVIER DE WEVER • Department of Radiotherapy and Nuclear Medicine, Laboratory of
Experimental Cancer Research, Ghent University Hospital, Ghent, Belgium
 Contributors xi

DANIEL WICKLEIN • Institute of Anatomy II: Experimental Morphology, University Medical

Center Hamburg-Eppendorf, Hamburg, Germany
CLARA M. YATES • School of Clinical and Experimental Medicine, College of Medical
and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK
WOLFRAM-HUBERTUS ZIMMERMANN • Department of Pharmacology, Georg-August-University,
Goettingen, Germany
 Chapter 1

In Vitro Invasion Assay Using Matrigel™: A Reconstituted

Basement Membrane Preparation
Debbie M.S. Hall and Susan A. Brooks

Abstract
Basement membranes, specialized extracellular matrices composed of collagens, laminins, and proteoglycans,
form thin, continuous sheetlike structures that separate epithelial tissues from adjacent connective tissues.
The crossing of basement membranes by cancer cells is a crucial aspect of metastasis—it must occur in
order that cancer cells can invade lymphatic or blood vessels during dissemination and also when they
penetrate into the target organ tissue where they will eventually colonize to form secondary tumors. The
assay system described in this chapter utilizes the solubilized basement membrane preparation Matrigel™
and measures the ability of cells to attach to the matrix, invade into and through the matrix, and migrate
towards a chemoattractant. It is technically straightforward and requires no specialist equipment and
provides a useful tool for assessing the invasive ability of cancer cells, exploring the functional role of
specific cell surface molecules/receptors in this process and screening for inhibitors of invasive ability, thus
contributing to current knowledge of the molecular events occurring during the invasive process.

Key words Basement membranes, Matrigel, Invasion assay, Extracellular matrix, Cell migration

1 Introduction

Basement membranes are specialized extracellular matrices compris-

ing several biological components including collagens, laminins,
and proteoglycans. They form thin continuous sheetlike structures
that separate epithelial tissues from the adjacent connective tissue
stroma; thus, they form barriers which block the passage of cells
and other macromolecules. The basement membranes become
permeable during tissue development and tissue repair and in order
to allow immune cells to reach the site, during inflammation [1].
The tumor invasion of basement membranes is thought to be
a crucial step in the complex “multistep event” that leads to suc-
cessful tumor metastasis (reviewed in ref. [2]). Tumor cells initially
cross basement membranes as they begin to invade the lymphatics
or vascular beds during their dissemination and also when they
penetrate into the target organ tissue where they will eventually

Miriam Dwek et al. (eds.), Metastasis Research Protocols, Methods in Molecular Biology, vol. 1070,
DOI 10.1007/978-1-4614-8244-4_1, © Springer Science+Business Media New York 2014

1
2 Debbie M.S. Hall and Susan A. Brooks

colonize to form secondary tumors. The penetration of the tumor

cells into basement membranes is thought to involve a series of
interdependent events. These include the initial attachment (adhesion)
of the cancer cells to components/receptors within the basement
membrane [3], degradation of the basement membranes (probably
through the action of proteolytic enzymes) [3–5], and, finally, the
migration of tumor cells into the target organ tissue in response to
specific chemotactic stimuli [6, 7].
In order to assess the invasive ability of tumor cells in vitro a
large number of systems have been developed which allow the
assessment of the capacity of tumor cells in vitro to invade basement
membranes. Several of these assays utilize basement membrane
extracts from tissues such as amnion [8], chick chorioallantoic
membranes [9], lens capsule [10], and bladder wall [11]. However,
the reproducibility of results using these substrata can often be
very difficult due to the inherent heterogeneity of the tissue prepa-
ration. Moreover, the process of extracting substrata from tissues is
often a long and technically challenging process. In order to
address these issues, reconstituted—and hence more homoge-
nous—extracellular matrices have been developed as alternative
substrata to investigate invasion in vitro.
One such example is Matrigel™—a solubilized basement
membrane preparation extracted from the Engelbreth-Holm-­
Swarm mouse sarcoma [12], a tumor rich in extracellular matrix
proteins. It mainly comprises laminin, collagen IV, heparan sulfate
proteoglycans, entactin, and nidogen, all components of human
basement membranes.
Matrigel™ is relatively simple to handle in the laboratory in
comparison to the substrata described previously. It can be dried
and then reconstituted onto membranes with 8 μm pores. These
Matrigel™-coated membranes act as barriers to tumor cell invasion
and subsequent migration through the pores in the membranes.
The invasion assay described in this chapter measures the ability
of cells to attach to the matrix, invade into and through the matrix,
and migrate towards a chemoattractant. These interdependent
steps are evidently crucial during the metastatic cascade. This assay
has been employed, for example, to determine whether a particular
class of aberrantly glycosylated glycoproteins are functionally
involved in cancer cell attachment to, or invasion through, base-
ment membrane components during metastasis [13].
The design of this assay permits easy experimental manipula-
tion when compared to other systems such as the Boyden chamber
assay. Moreover, there is no need to buy any expensive specialized
equipment. In addition, the assay described in this chapter is
carried out under sterile conditions, therefore allowing cells to be
recovered and utilized in subsequent studies. This might prove
­difficult if basement membrane components extracted from fresh
tissue were used.
 In Vitro Invasion Assay Using Matrigel™… 3

This assay system enables the researcher to screen inhibitors

seeking those which may alter the invasive phenotype of the tumor
cells, thus contributing to current knowledge of the molecular
events occurring during the invasive process.
Before commencing any detailed research using this assay, it is
first of all important to carry out empirical studies in order to opti-
mize the conditions the experiments will be performed under.
These initial studies should be carried out in order to determine
the optimal concentration of Matrigel™ suitable for use in the
experiments. If the concentration used is too low then it will not
provide a realistic barrier to differentiate between invasive and
noninvasive cells. Conversely, if the Matrigel™ used is too highly
concentrated, then it will be difficult for even very invasive cells to
penetrate the barrier that it provides. We suggest trying several
dilutions of the Matrigel™ solution.
It is equally important that the researcher determines the
optimum number of cells for use in each of the assays. This is likely
to be different for different cell lines or types. Using too many
cells will result in “clumping,” and this will obviously give
erroneous results. We suggest screening a range of different cell
concentrations.
This part of the work takes some time; however, once com-
pleted, the assays can be done relatively quickly and can be adapted
according to the researcher’s needs.

2 Materials

2.1 Preparation 1. Falcon multiwell companion plates (available as 6, 12, or

of the Matrigel™ 24 well).
Chambers 2. Falcon cell culture inserts with 8 μm pores (also available in 6-,
12-, or 24-well format).
3. Matrigel™ matrix solution. Stable for at least 9 months when
stored at −20 °C.
4. Sterile 7 ml Bijou bottles.
5. Sterile pipette tips.
6. Standard cell culture medium (e.g., DMEM) without fetal
calf serum.
7. Pair of sterile forceps.

2.2 Preparation 1. Cell line(s) to be analyzed (see Note 1).

of the Cells 2. Standard cell culture medium without fetal calf serum.
3. Phosphate-buffered saline (PBS) solution: Dissolve 8 g of
sodium chloride, 0.2 g of potassium chloride, 1.44 g of sodium
phosphate (bi-basic), and 0.24 g potassium phosphate in 800 ml
4 Debbie M.S. Hall and Susan A. Brooks

of distilled water. Adjust the pH to 7.2 and then adjust the

volume to 1 l with distilled water. Dispense into convenient vol-
umes and sterilize by autoclaving. Store at room temperature.
4. Ethylenediaminetetraacetate (EDTA) solution: Add 0.1 g of
EDTA to 500 ml of PBS (see above). Add sodium hydroxide
to adjust the pH to 8.0 and to allow the EDTA to dissolve.
Dispense into convenient volumes and sterilize by autoclaving.
Store at room temperature.
5. 0.05 % Trypsin–0.02 % w/v EDTA solution.
6. Standard cell culture medium with 10 % v/v heat-inactivated
fetal calf serum.
7. Sterile 15 ml centrifuge tubes.
8. Standard cell culture medium with 0.1 % w/v sterile filtered
bovine serum albumin (BSA) (fraction V).
9. Hemocytometer.
10. Trypan blue solution: 0.25 % w/v trypan blue in PBS, filter
sterilized. Store at room temperature. Stable for several years.

2.3 Coomassie Blue 1. Coomassie brilliant blue solution: Dissolve 0.25 g Coomassie
Staining to Check the Brilliant Blue R-250 in a solution of 50 ml methanol, 10 ml
Matrigel™ Coating acetic acid, and 40 ml distilled water. Filter sterilize.
2. Destain solution: Mix 5 ml methanol, 7.5 ml acetic acid, and
80 ml of distilled water. Filter sterilize.

2.4 Matrigel™ 1. Standard cell culture medium with 10 % v/v fetal calf serum.
Invasion Assay 2. Fixative: 4 % v/v formal saline solution: Dissolve 4.25 g sodium
chloride in 500 ml of a 4 % v/v formaldehyde solution in
distilled water. Alternatively, use ice-cold 100 % methanol.
3. 6-, 12-, or 24-well multiwell plates or companion plates (need
not be sterile).
4. Sterile cotton swabs.
5. Standard cell culture medium without fetal calf serum.
6. Pair of sterile forceps.
7. Trypsin–EDTA solution (as item 5, Subheading 2.2).
8. Mayer’s hematoxylin solution.
9. 1 % w/v aqueous eosin solution.
10. 70 % v/v ethanol or industrial methylated spirit in distilled
water.
11. 100 % ethanol or industrial methylated spirit.
12. Xylene.
13. Scalpel (No. 11 blade recommended).
14. Pair of forceps (need not be sterile).
 In Vitro Invasion Assay Using Matrigel™… 5

15. Microscope slides and coverslips.

16. Xylene-based mounting medium (e.g., Depex).
17. 10 × 10 eye-piece graticule.

3 Methods

3.1 Preparation 1. Defrost the Matrigel™ solution for at least 6 h on ice in a

of the Matrigel™ refrigerator. We recommend overnight as this is more conve-
Chambers nient (see Note 2).
2. Precool all plates, inserts, sterile pipettes, and 7 ml Bijou
bottles in a refrigerator overnight (see Note 3).
N.B. Unless otherwise indicated, all the following procedures
should be conducted under sterile conditions.
3. Shake the bottle to thoroughly mix the Matrigel™ solution.
Dilute the defrosted Matrigel™ into a variety of concentrations
for use in the empirical studies as described in the introduc-
tion. Start with concentrations of 1.2, 0.6, and 0.3 mg/ml
diluted in cell culture medium without fetal calf serum. Pipette
5 ml aliquots into 7 ml Bijou bottles, refreeze, and store those
not to be used immediately at −20 °C. When defrosting for
later experiments, do so as described previously in step 1,
Subheading 3.1.
4. Using sterile forceps, remove the precooled inserts from their
packaging and carefully place into the housing on the pre-
cooled companion plates.
5. In a laminar flow hood, at room temperature, carefully pipette
the diluted Matrigel™ solution on top of the insert and gently
rotate the insert to ensure that the entire filter is coated (for a
6-well format use 300 μl; for a 12-well use 100 μl; and for a
24-well use 40 μl of Matrigel™ solution). Very carefully over-
lay the Matrigel™ with sterile double-distilled water (for a
6-well format use 200 μl; for a 12-well use 100 μl; and for a
24-well use 50 μl distilled water) using a sterile pipette. This
stage should be performed in a minimum of triplicate wells.
6. Control inserts should also be prepared. These are simply
inserts without the Matrigel™ layer. In order to prepare these,
simply place the empty inserts into the companion plate and
leave in the laminar flow hood alongside the inserts which are
being prepared with Matrigel™. Follow all other relevant steps.
7. Leave the Matrigel™ layer to air-dry (in the laminar flow hood
at room temperature under occasional UV light to maintain
sterile conditions). This stage usually takes around 1–2 days.
8. Rehydrate the dried Matrigel™ layer by adding warm (37 °C)
cell culture medium without fetal calf serum (for 6-well format,
6 Debbie M.S. Hall and Susan A. Brooks

use 600 μl; for 12-well, use 200 μl; and for 24-well, use 75 μl
culture medium) to the plates. Allow the Matrigel™ layer to
rehydrate for around 2 h in a cell culture incubator at 37 °C
(see Note 4).

3.2 Preparation 1. Wash cultured cell monolayers as follows: Pipette cell culture
of the Cells medium without fetal calf serum onto the monolayer, and
gently rock the flask from side to side so that the entire cell
monolayer is covered. Discard the medium. Repeat three times.
2. Wash the cultured cell monolayer once, as described in step 1,
Subheading 3.2, but this time using PBS. Use 4 ml for a 75 cm2
flask, and 2 ml for a 25 cm2 flask. Aspirate and discard the PBS.
3. Incubate the cells with EDTA solution (use the same volumes
as for PBS, see step 2, Subheading 3.2) for around 10 min in a
laminar flow hood.
4. Aspirate and discard the EDTA solution and pipette on the
trypsin–EDTA solution (2.5 ml for a 75 cm2 flask and 1 ml for
a 25 cm2 flask). Tighten the cap on the flask and gently swirl
the solution over the surface of the cells. Place the flask of cells
into a cell culture incubator. After around 30 s to 1 min, pipette
off any excess solution and monitor the progress of the cells
under an inverted microscope (see Note 5). Tap the flask
sharply to loosen the cells from the surface. As soon as the
majority of the cells are detached from the flask stop the action
of the trypsin by the addition of complete cell culture medium
with 10 % v/v fetal calf serum (5 ml in a 75 cm2 flask and 2 ml
in a 25 cm2 flask).
5. Transfer the contents of the flask into a 15 ml sterile centrifuge
tube and pellet the cells at 1,100 × g for 5 min. Pipette off the
medium without disturbing the cell pellet. Gently tap the cen-
trifuge tube against the bench to loosen the cell pellet and
resuspend in 5 ml of cell culture medium without fetal calf
serum. Repeat twice more. The cell pellet should be re-­
suspended in a final volume of 2.5 ml cell culture medium with
0.1 % w/v BSA.
6. Count the cell suspension using a hemocytometer (see Note 6).
7. Assess cell viability using trypan blue. Trypan blue is excluded
from living cells but stains dead/damaged cells blue. To do
this, mix 50 μl of trypan blue solution with 50 μl of cell suspen-
sion. Transfer the mixture to a hemocytometer and observe
under the microscope. The total number of viable cells can be
calculated directly as follows:
Total number of viable cells
× 2 × 10 4 = number of viable cells / mL.
Number of squares counted
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 Immunology - Term Paper
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Prepared by: Prof. Brown

Date: July 28, 2025

Section 1: Best practices and recommendations

Learning Objective 1: Historical development and evolution
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 2: Theoretical framework and methodology
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Comparative analysis and synthesis
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 4: Statistical analysis and interpretation
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Learning Objective 5: Historical development and evolution
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 5: Diagram/Chart/Graph]
Note: Experimental procedures and results
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 6: Diagram/Chart/Graph]
Important: Comparative analysis and synthesis
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Theoretical framework and methodology
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Practical applications and examples
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Experimental procedures and results
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 10: Diagram/Chart/Graph]
Module 2: Fundamental concepts and principles
Definition: Critical analysis and evaluation
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Problem-solving strategies and techniques
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Key terms and definitions
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 13: Study tips and learning strategies
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 14: Diagram/Chart/Graph]
Note: Fundamental concepts and principles
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 15: Diagram/Chart/Graph]
Definition: Practical applications and examples
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Key Concept: Experimental procedures and results
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 17: Practical applications and examples
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 18: Diagram/Chart/Graph]
Example 18: Research findings and conclusions
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 19: Diagram/Chart/Graph]
Remember: Interdisciplinary approaches
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Review 3: Ethical considerations and implications
Example 20: Best practices and recommendations
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Research findings and conclusions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 22: Experimental procedures and results
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 23: Practical applications and examples
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Historical development and evolution
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Study tips and learning strategies
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 26: Diagram/Chart/Graph]
Important: Fundamental concepts and principles
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Practice Problem 27: Case studies and real-world applications
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 28: Literature review and discussion
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 29: Interdisciplinary approaches
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Module 4: Historical development and evolution
Example 30: Theoretical framework and methodology
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 31: Research findings and conclusions
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 32: Diagram/Chart/Graph]
Example 32: Critical analysis and evaluation
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 33: Experimental procedures and results
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Research findings and conclusions
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 35: Ethical considerations and implications
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 36: Diagram/Chart/Graph]
Definition: Historical development and evolution
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 37: Diagram/Chart/Graph]
Remember: Historical development and evolution
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 38: Theoretical framework and methodology
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Experimental procedures and results
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Discussion 5: Study tips and learning strategies
Definition: Problem-solving strategies and techniques
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 41: Interdisciplinary approaches
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 42: Diagram/Chart/Graph]
Note: Case studies and real-world applications
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 43: Diagram/Chart/Graph]
Practice Problem 43: Theoretical framework and methodology
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Research findings and conclusions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 45: Diagram/Chart/Graph]
Remember: Critical analysis and evaluation
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Experimental procedures and results
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Assessment criteria and rubrics
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Statistical analysis and interpretation
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Study tips and learning strategies
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Part 6: Research findings and conclusions
Practice Problem 50: Critical analysis and evaluation
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 51: Diagram/Chart/Graph]
Example 51: Practical applications and examples
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
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