Table Of ContentCell Growth, Differentiation and Senescence
The Practical Approach Series
SERIES EDITOR
B. D. HAMES
Department of Biochemistry and Molecular Biology
University of Leeds, Leeds LS2 9JT, UK
See also the Practical Approach web site at http://www.oup.co.uk/PAS
* indicates new and forthcoming titles
Affinity Chromatography if Cell Separation
Affinity Separations Cellular Calcium
Anaerobic Microbiology Cellular Interactions in
Development
Animal Cell Culture (2nd
edition) Cellular Neurobiology
Animal Virus Pathogenesis * Chrornatin
Antibodies I and II * Chromosome Structural
Antibody Engineering Analysis
Clinical Immunology
* Antisense Technology
Complement
Applied Microbial
Physiology if Crystallization of Nucleic
Acids and Proteins (2nd
Basic Cell Culture
edition)
Behavioural Neuroscience
Cytokines (2nd edition)
Bioenergetics
The Cytoskeleton
Biological Data Analysis
Diagnostic Molecular
Biomechanics - Materials
Pathology I and II
Biomechanics — Structures and
DNA and Protein Sequence
Systems
Analysis
Biosensors
DNA Cloning 1: Core
Carbohydrate Analysis (2nd Techniques (2nd edition)
edition)
DNA Cloning 2: Expression
Cell-Cell Interactions Systems (2nd edition)
The Cell Cycle
DNA Cloning 3: Complex
Cell Growth and Apoptosis Genomes (2nd edition)
DNA Cloning 4: Mammalian Human Genetic Disease
Systems (2nd edition) Analysis
* Drosophila (2nd edition) if Immobilized Biomolecules in
Electron Microscopy in Analysis
Biology Immunochemistry 1
Electron Microscopy in Immunochemistry 2
Molecular Biology
Immunocytochemistry
Electrophysiology
* In Situ Hybridization (2nd
Enzyme Assays edition)
Epithelial Cell Culture lodinated Density Gradient
Essential Developmental Media
Biology Essential Molecular Ion Channels
Biology I and I
* Light Microscopy (2nd edition)
* Eukaryotic DNA Replication
Lipid Modification of Proteins
Experimental Neuroanatomy
Lipoprotein Analysis
Extracellular Matrix
Liposomes
Flow Cytometry (2nd edition)
Mammalian Cell
Free Radicals
Biotechnology
Gas Chromatography
Medical Parasitology
Gel Electrophoresis of Nucleic
Medical Virology
Acids (2nd edition)
MHC Volumes 1 and 2
* Gel Electrophoresis of Proteins
* Molecular Genetic Analysis of
(3rd edition)
Populations (2nd edition)
Gene Probes 1 and 2
Molecular Genetics of Yeast
Gene Targeting
Molecular Imaging in
Gene Transcription
Neuroscience
if Genome Mapping
Molecular Neurobiology
Glycobiology
Molecular Plant Pathology I
* Growth Factors and
and II
Receptors
Molecular Virology
Haemopoiesis
Monitoring Neuronal Activity
Histocompatibility Testing
Mutagenicity Testing
HIV Volumes l and 2
* Mutation Detection
* HPLC of Macromolecules (2nd
Neural Cell Culture
edition)
Neural Transplantation
Human Cytogenetics I and II
(2nd edition) Neurochemistry (2nd edition)
Neuronal Cell Lines if Protein Expression Vol 1
NMR of Biological if Protein Expression Vol 2
Macromolecules
Protein Engineering
Non-isotopic Methods in
Protein Function (2nd edition)
Molecular Biology
Protein Phosphorylation
Nucleic Acid Hybridisation
Protein Purification
Oligonucleotides and
Applications
Analogues
Protein Purification Methods
Oligonucleotide Synthesis
Protein Sequencing
PCR 1
Protein Structure
PCR 2
(2nd edition)
* PCR 3: PCR In Situ
Protein Structure Prediction
Hybridization
Protein Targeting
Peptide Antigens
Proteolytic Enzymes
Photosynthesis: Energy
Trans duction Pulsed Field Gel
Electrophoresis
Plant Cell Biology
Plant Cell Culture (2nd edition) RNA Processing I and II
Plant Molecular Biology * RNA-Protein Interactions
Plasmids (2nd edition) Signalling by Inositides
Platelets Subcellular Fractionation
Postimplantation Mammalian Signal Transduction
Embryos if Transcription Factors (2nd
Preparative Centrifugation edition)
Protein Blotting Tumour Immunobiology
Cell Growth,
Differentiation and
Senescence
A Practical Approach
Edited by
GEORGE P. STUDZINSKI
Department of Pathology and Laboratory Medicine,
UMD—New Jersey Medical School,
Newark, N.J., USA
OXFORD
UNIVERSITY PRESS
OXFORD
UNIVERSITY PRESS
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Library of Congress Cataloging in Publication Data
Cell growth, differentiation, and senescense : a practical approach /
edited by George P. Studzinski.
(Practical approach series ; 215)
Includes bibliographical references and index.
1. Cells—Growth—Research Laboratory manuals. 2. Cell
differentiation Laboratory manuals. 3. Cell death Laboratory
manuals. I. Studzinski, George P. II. Series.
QH604.7.C447 1999 571.8-dc21 99-33469
ISBN 0 19 963 769 5 (Hbk)
0 19 963 768 7 (Pbk)
Typeset by Footnote Graphics,
Warminster, Wilts
Printed in Great Britain by Information Press, Ltd,
Eynsham, Oxon.
Preface
This volume presents a variety of approaches to the study of mammalian cell
growth, ranging from detailed presentations of the recent modifications of the
established general procedures for measurement of growth and cytotoxicity,
through examples of the examination of the growth signalling pathway, to the
establishment of growth cessation through differentiation or senescence. The
conceptual underpinnings of each approach are provided, together with the
details of procedures found most useful in each author's laboratory and guide-
lines for interpretation of the expected results.
Current studies of growth-associated phenomena go well beyond an enu-
meration of cell proliferation and while it is not possible to cover every facet of
this rapidly advancing field, examples are included of such advanced tech-
niques as the assessment of cell cycle checkpoints, detection of oncogenes, and
the examination of the nuclear architecture of growing cells.
The negative aspects of cell growth are perhaps of even greater importance
than cell proliferation itself, since this is the focus of intense efforts to control
human diseases such as cancer. In this vein, the volume presents several
approaches to studies of controlled cessation of cell proliferation, through
induction of differentiation or by evolving senescence. The principal focus is
on human cells. It should be also noted that an important aspect of control of
cell proliferation is not included here, since it is a subject of a companion pub-
lication, Apoptosis: A Practical Approach.
The authors and the staff of OUP have made a concerted effort to provide a
truly practical compendium on how to study cell growth; it is sincerely hoped
that this labour will fulfil the needs of beginning as well as experienced investi-
gators, and help to inspire their efforts as well as to provide specific guidance.
New Jersey G.P.S.
1999
Contents
List of Contributors xvii
Abbreviations xxi
1. Selection of methods for measuring
proliferation 1
Robert Wieder
1. Introduction 1
Defining parameters of proliferation 1
2. Cells in tissue culture 4
Counting cell numbers 4
Measuring DNA content 8
Measuring the rate of mitosis 13
Measuring DNA synthesis 15
Measuring active metabolism as a reflection of viable cell number:
the MTT assay 18
Measuring the collective cell volume 19
3. Determining proliferation in vivo 20
Measuring DNA synthesis 20
4. Cells and tissue samples obtained from human patients
or animals 26
Determining the mitotic index 27
Measuring cellular DNA content 27
Nuclear antigens associated with proliferation: PCNA, Ki67, and
AgNOR 27
References 30
2. Cell growth and cytotoxicity assays 33
Philip Skehan
1. Introduction 33
2. Growth and cytotoxicity assays 33
3. Quantifying cell growth 34
Experimental design 34
Growth rate and doubling time 35
Potency 35
Efficacy 36
Contents
4. Cell cultures 36
Seeding density 36
Dissociation and recovery 36
Drug solubilization 37
Assay duration 37
Control wells on every plate 37
5. Dye-binding assays 37
Optimizing and validating dye-binding assays 38
Protein and biomass stains 39
6. Metabolic impairment assays 45
Neutral red cell viability assay 45
MTT redox assay 46
AlamarBlue (ALB) oxidation-reduction assay 48
Cellular ATP assay 51
7. Membrane integrity assays 52
Fluorescein diacetate (FDA) 52
8. Survivorship assays 53
Adherent versus non-adherent cells 54
Long-term recovery (LTR) assay 54
Colony-forming efficiency on tissue culture plastic 56
Colony-forming efficiency in soft agar 57
References 59
3. Cell growth and kinetics in multicell
spheroids 61
Ralph E. Durand
1. Introduction 61
2. Growth of spheroids 61
Options 61
Example: the V79 spheroid system 64
3. Special features of spheroids 65
Metabolite and catabolite gradients 65
Viability in spheroid cell subpopulations 66
4. Unexpected features of spheroids 66
Genetic instability 66
Aneuploidy—a response to 'architecture' 68
5. Cell kinetics in spheroids 68
Problems 68
Approaches 68
Example: kinetics in V79 spheroids 71
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