,Methods in Molecular Biology™
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For other titles published in this series, go to
www.springer.com/series/7651
,
,Mammalian Cell Viability
Methods and Protocols
Edited by
Martin J. Stoddart
AO Research Institute Davos, Davos Platz, Switzerland
,Editor
Martin J. Stoddart, Ph.D.
AO Research Institute Davos
Davos Platz, Switzerland
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-61779-107-9 e-ISBN 978-1-61779-108-6
DOI 10.1007/978-1-61779-108-6
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011925550
© Springer Science+Business Media, LLC 2011
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
While the advice and information in this book are believed to be true and accurate at the date of going to press, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may
be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
Humana Press is part of Springer Science+Business Media (www.springer.com)
,Preface
Viability of cells is one of the most fundamental measurements made during studies in cell
biology. Whether the question is one of basic cell survival, or whether it is being used to
correlate cell number to some other factors such as matrix synthesis, an estimate of cell
viability is universally required. The specific method used will greatly influence the inter-
pretation of the data. While many viability methods have been used for decades, there
have been recent developments which offer increased sensitivity, throughput, and specific-
ity. The particular type of cell death, apoptotic or necrotic, is becoming increasingly
important. This requires multiplexing of methods, or methods that are able to distinguish
between the different cell states. This book aims to bring together a wide array of methods
in order to assist the reader to determine which is most suitable for them. Certain meth-
ods will provide information about the population as a whole, while others determine
viability on single cell level. In some cases, it may also be important to realize that the
method, while producing an answer, is not suitable for the application applied. It is hoped
that the pros and cons of each method will become clear. Many methods have been devised for
monolayer cell culture, and although they can often be translated into a three-dimensional
system, care must be taken to limit artifacts.
Multiplexing assays is one mechanism by which many sets of data can be obtained from
a small number of samples. Owing to the wide array of various viability assays, with the
option for both colorimetric and fluorescent measurements, the potential combinations
are endless and could not be covered within a book. While many chapters within this book
employ more than one assay, two chapters specifically dealing with 96-well multiplexing
are provided in order to illustrate practical examples. These can also form a basis which can
be modified and utilized with other assays. A number of kits are now commercially avail-
able for both single assays and multiplexing, and often the underlying technology of the
different kits used is comparable. It is for this reason a good understanding of the reagent
used, and how it functions, is crucial when accurately interpreting the data obtained.
This book describes methods from the most basic level, which can be performed in
any laboratory, to more complex methods which require specialist pieces of equipment.
Initially, the chapters are focused on methods for monolayer and suspension cells; later
chapters describe methods for determining viability within tissue sections and three-
dimensional culture systems. Finally, methods requiring highly specialized equipment are
described in order to explain what is possible. The last chapter aims to provide some guid-
ance as to how automated image analysis can reduce time and inconsistency of quantifying
large numbers of images of live or dead cells.
In preparing Mammalian Cell Viability: Methods and Protocols, the aim has been to
produce a self-contained laboratory manual which is useful for both experienced research-
ers and those new to the field. I hope that everyone can learn something from this book.
Finally, I wish to thank all the contributing authors, as well as John Walker and the staff at
Humana Press for seeing this project through.
Davos Platz, Switzerland Martin J. Stoddart
v
,
,Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Cell Viability Assays: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Martin J. Stoddart
2 Cell Viability Analysis Using Trypan Blue: Manual and Automated Methods . . . . 7
Kristine S. Louis and Andre C. Siegel
3 Estimation of Cell Number Based on Metabolic Activity:
The MTT Reduction Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
László Kupcsik
4 WST-8 Analysis of Cell Viability During Osteogenesis of Human
Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Martin J. Stoddart
5 Assessment of Cell Proliferation with Resazurin-Based Fluorescent Dye . . . . . . . . 27
Ewa M. Czekanska
6 The xCELLigence System for Real-Time and Label-Free
Monitoring of Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Ning Ke, Xiaobo Wang, Xiao Xu, and Yama A. Abassi
7 Analysis of Tumor and Endothelial Cell Viability and Survival
Using Sulforhodamine B and Clonogenic Assays . . . . . . . . . . . . . . . . . . . . . . . . . 45
Caroline Woolston and Stewart Martin
8 Annexin V/7-AAD Staining in Keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Maya Zimmermann and Norbert Meyer
9 Measurement of Caspase Activity: From Cell Populations
to Individual Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Gabriela Paroni and Claudio Brancolini
10 Rapid Quantification of Cell Viability and Apoptosis in B-Cell
Lymphoma Cultures Using Cyanine SYTO Probes . . . . . . . . . . . . . . . . . . . . . . . 81
Donald Wlodkowic, Joanna Skommer, and Zbigniew Darzynkiewicz
11 Multiplexing Cell Viability Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Helga H.J. Gerets, Stéphane Dhalluin, and Franck A. Atienzar
12 Cytotoxicity Testing: Measuring Viable Cells, Dead Cells,
and Detecting Mechanism of Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Terry L. Riss, Richard A. Moravec, and Andrew L. Niles
13 Fluorescein Diacetate for Determination of Cell Viability
in 3D Fibroblast–Collagen–GAG Constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Heather M. Powell, Alexis D. Armour, and Steven T. Boyce
vii
,viii Contents
14 Confocal Imaging Protocols for Live/Dead Staining
in Three-Dimensional Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Benjamin Gantenbein-Ritter, Christoph M. Sprecher,
Samantha Chan, Svenja Illien-Jünger, and Sibylle Grad
15 Viability Assessment of Osteocytes Using Histological Lactate
Dehydrogenase Activity Staining on Human Cancellous Bone Sections . . . . . . . . 141
Katharina Jähn and Martin J. Stoddart
16 Measuring Glutamate Receptor Activation-Induced Apoptotic
Cell Death in Ischemic Rat Retina Using the TUNEL Assay . . . . . . . . . . . . . . . . 149
Won-Kyu Ju and Keun-Young Kim
17 Exploiting the Liberation of Zn2+ to Measure Cell Viability . . . . . . . . . . . . . . . . . 157
Christian J. Stork and Yang V. Li
18 Noninvasive Bioluminescent Quantification of Viable
Stem Cells in Engineered Constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Karim Oudina, Adeline Cambon-Binder,
and Delphine Logeart-Avramoglou
19 Raman Micro-Spectroscopy as a Non-invasive Cell Viability Test . . . . . . . . . . . . . 179
Sophie Verrier, Alina Zoladek, and Ioan Notingher
20 Closed Ampoule Isothermal Microcalorimetry for Continuous
Real-Time Detection and Evaluation of Cultured Mammalian
Cell Activity and Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Olivier Braissant and Alma U. “Dan” Daniels
21 Digital Image Processing of Live/Dead Staining . . . . . . . . . . . . . . . . . . . . . . . . . 209
Pieter Spaepen, Sebastian De Boodt, Jean-Marie Aerts,
and Jos Vander Sloten
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
, Contributors
Yama A. Abassi • ACEA Biosciences Inc., San Diego, CA, USA
Jean-Marie Aerts • Division M3-BIORES: Measure, Model & Manage Bioresponses,
Katholieke Universiteit Leuven, Heverlee, Belgium
Alexis D. Armour • Department of Surgery, University of Missouri, Columbia,
MO, USA
Franck A. Atienzar • Investigative Non-Clinical Safety, Non-Clinical Development,
UCB Pharma SA, Braine-l’Alleud, Belgium
Sebastian De Boodt • Division M3-BIORES: Measure, Model & Manage
Bioresponses, Katholieke Universiteit Leuven, Heverlee, Belgium
Steven T. Boyce • Department of Surgery, University of Cincinnati, Cincinnati,
OH, USA; Department of Biomedical Engineering, University of Cincinnati,
Cincinnati, OH, USA; Research Department, Shriners Hospitals for Children,
Cincinnati, OH, USA
Olivier Braissant • Laboratory of Biomechanics & Biocalorimetry, Coalition
for Clinical Morphology & Biomedical Engineering, University of Basel,
Basel, Switzerland
Claudio Brancolini • Dipartimento di Scienze e Tecnologie Biomediche,
Università degli Studi di Udine, Udine, Italy
Adeline Cambon-Binder • Laboratoire de Bio-ingéniérie et Biomécanique
Ostéo-articulaires, UMR, CNRS, 7052, Paris, France
Samantha Chan • ARTORG, Center for Biomedical Engineering Research,
Institute for Surgical Technology and Biomechanics, University of Bern,
Bern, Switzerland
Ewa M. Czekanska • AO Research Institute, Davos Platz, Switzerland
Alma U. “Dan” Daniels • Laboratory of Biomechanics & Biocalorimetry,
Coalition for Clinical Morphology & Biomedical Engineering, University of Basel,
Basel, Switzerland
Zbigniew Darzynkiewicz • Brander Cancer Research Institute, New York
Medical College, Valhalla, NY, USA
Stéphane Dhalluin • Investigative Non-Clinical Safety, Non-Clinical Development,
UCB Pharma SA, Braine-l’Alleud, Belgium
Benjamin Gantenbein-Ritter • ARTORG, Center for Biomedical Engineering
Research, Institute for Surgical Technology and Biomechanics, University of Bern,
Bern, Switzerland
Helga H.J. Gerets • Investigative Non-Clinical Safety, Non-Clinical Development,
UCB Pharma SA, Braine-l’Alleud, Belgium
Sibylle Grad • AO Research Institute, Davos Platz, Switzerland
Svenja Illien-Jünger • AO Research Institute, Davos Platz, Switzerland;
Department of Orthopaedics, Mount Sinai Medical Centre, New York, USA
Katharina Jähn • School of Dentistry Department of Oral Biology University
Missouri, Kansas City, Missouri, USA
ix
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For other titles published in this series, go to
www.springer.com/series/7651
,
,Mammalian Cell Viability
Methods and Protocols
Edited by
Martin J. Stoddart
AO Research Institute Davos, Davos Platz, Switzerland
,Editor
Martin J. Stoddart, Ph.D.
AO Research Institute Davos
Davos Platz, Switzerland
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-61779-107-9 e-ISBN 978-1-61779-108-6
DOI 10.1007/978-1-61779-108-6
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011925550
© Springer Science+Business Media, LLC 2011
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of
the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
While the advice and information in this book are believed to be true and accurate at the date of going to press, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may
be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
Humana Press is part of Springer Science+Business Media (www.springer.com)
,Preface
Viability of cells is one of the most fundamental measurements made during studies in cell
biology. Whether the question is one of basic cell survival, or whether it is being used to
correlate cell number to some other factors such as matrix synthesis, an estimate of cell
viability is universally required. The specific method used will greatly influence the inter-
pretation of the data. While many viability methods have been used for decades, there
have been recent developments which offer increased sensitivity, throughput, and specific-
ity. The particular type of cell death, apoptotic or necrotic, is becoming increasingly
important. This requires multiplexing of methods, or methods that are able to distinguish
between the different cell states. This book aims to bring together a wide array of methods
in order to assist the reader to determine which is most suitable for them. Certain meth-
ods will provide information about the population as a whole, while others determine
viability on single cell level. In some cases, it may also be important to realize that the
method, while producing an answer, is not suitable for the application applied. It is hoped
that the pros and cons of each method will become clear. Many methods have been devised for
monolayer cell culture, and although they can often be translated into a three-dimensional
system, care must be taken to limit artifacts.
Multiplexing assays is one mechanism by which many sets of data can be obtained from
a small number of samples. Owing to the wide array of various viability assays, with the
option for both colorimetric and fluorescent measurements, the potential combinations
are endless and could not be covered within a book. While many chapters within this book
employ more than one assay, two chapters specifically dealing with 96-well multiplexing
are provided in order to illustrate practical examples. These can also form a basis which can
be modified and utilized with other assays. A number of kits are now commercially avail-
able for both single assays and multiplexing, and often the underlying technology of the
different kits used is comparable. It is for this reason a good understanding of the reagent
used, and how it functions, is crucial when accurately interpreting the data obtained.
This book describes methods from the most basic level, which can be performed in
any laboratory, to more complex methods which require specialist pieces of equipment.
Initially, the chapters are focused on methods for monolayer and suspension cells; later
chapters describe methods for determining viability within tissue sections and three-
dimensional culture systems. Finally, methods requiring highly specialized equipment are
described in order to explain what is possible. The last chapter aims to provide some guid-
ance as to how automated image analysis can reduce time and inconsistency of quantifying
large numbers of images of live or dead cells.
In preparing Mammalian Cell Viability: Methods and Protocols, the aim has been to
produce a self-contained laboratory manual which is useful for both experienced research-
ers and those new to the field. I hope that everyone can learn something from this book.
Finally, I wish to thank all the contributing authors, as well as John Walker and the staff at
Humana Press for seeing this project through.
Davos Platz, Switzerland Martin J. Stoddart
v
,
,Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Cell Viability Assays: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Martin J. Stoddart
2 Cell Viability Analysis Using Trypan Blue: Manual and Automated Methods . . . . 7
Kristine S. Louis and Andre C. Siegel
3 Estimation of Cell Number Based on Metabolic Activity:
The MTT Reduction Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
László Kupcsik
4 WST-8 Analysis of Cell Viability During Osteogenesis of Human
Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Martin J. Stoddart
5 Assessment of Cell Proliferation with Resazurin-Based Fluorescent Dye . . . . . . . . 27
Ewa M. Czekanska
6 The xCELLigence System for Real-Time and Label-Free
Monitoring of Cell Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Ning Ke, Xiaobo Wang, Xiao Xu, and Yama A. Abassi
7 Analysis of Tumor and Endothelial Cell Viability and Survival
Using Sulforhodamine B and Clonogenic Assays . . . . . . . . . . . . . . . . . . . . . . . . . 45
Caroline Woolston and Stewart Martin
8 Annexin V/7-AAD Staining in Keratinocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Maya Zimmermann and Norbert Meyer
9 Measurement of Caspase Activity: From Cell Populations
to Individual Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Gabriela Paroni and Claudio Brancolini
10 Rapid Quantification of Cell Viability and Apoptosis in B-Cell
Lymphoma Cultures Using Cyanine SYTO Probes . . . . . . . . . . . . . . . . . . . . . . . 81
Donald Wlodkowic, Joanna Skommer, and Zbigniew Darzynkiewicz
11 Multiplexing Cell Viability Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Helga H.J. Gerets, Stéphane Dhalluin, and Franck A. Atienzar
12 Cytotoxicity Testing: Measuring Viable Cells, Dead Cells,
and Detecting Mechanism of Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Terry L. Riss, Richard A. Moravec, and Andrew L. Niles
13 Fluorescein Diacetate for Determination of Cell Viability
in 3D Fibroblast–Collagen–GAG Constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Heather M. Powell, Alexis D. Armour, and Steven T. Boyce
vii
,viii Contents
14 Confocal Imaging Protocols for Live/Dead Staining
in Three-Dimensional Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Benjamin Gantenbein-Ritter, Christoph M. Sprecher,
Samantha Chan, Svenja Illien-Jünger, and Sibylle Grad
15 Viability Assessment of Osteocytes Using Histological Lactate
Dehydrogenase Activity Staining on Human Cancellous Bone Sections . . . . . . . . 141
Katharina Jähn and Martin J. Stoddart
16 Measuring Glutamate Receptor Activation-Induced Apoptotic
Cell Death in Ischemic Rat Retina Using the TUNEL Assay . . . . . . . . . . . . . . . . 149
Won-Kyu Ju and Keun-Young Kim
17 Exploiting the Liberation of Zn2+ to Measure Cell Viability . . . . . . . . . . . . . . . . . 157
Christian J. Stork and Yang V. Li
18 Noninvasive Bioluminescent Quantification of Viable
Stem Cells in Engineered Constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Karim Oudina, Adeline Cambon-Binder,
and Delphine Logeart-Avramoglou
19 Raman Micro-Spectroscopy as a Non-invasive Cell Viability Test . . . . . . . . . . . . . 179
Sophie Verrier, Alina Zoladek, and Ioan Notingher
20 Closed Ampoule Isothermal Microcalorimetry for Continuous
Real-Time Detection and Evaluation of Cultured Mammalian
Cell Activity and Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Olivier Braissant and Alma U. “Dan” Daniels
21 Digital Image Processing of Live/Dead Staining . . . . . . . . . . . . . . . . . . . . . . . . . 209
Pieter Spaepen, Sebastian De Boodt, Jean-Marie Aerts,
and Jos Vander Sloten
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
, Contributors
Yama A. Abassi • ACEA Biosciences Inc., San Diego, CA, USA
Jean-Marie Aerts • Division M3-BIORES: Measure, Model & Manage Bioresponses,
Katholieke Universiteit Leuven, Heverlee, Belgium
Alexis D. Armour • Department of Surgery, University of Missouri, Columbia,
MO, USA
Franck A. Atienzar • Investigative Non-Clinical Safety, Non-Clinical Development,
UCB Pharma SA, Braine-l’Alleud, Belgium
Sebastian De Boodt • Division M3-BIORES: Measure, Model & Manage
Bioresponses, Katholieke Universiteit Leuven, Heverlee, Belgium
Steven T. Boyce • Department of Surgery, University of Cincinnati, Cincinnati,
OH, USA; Department of Biomedical Engineering, University of Cincinnati,
Cincinnati, OH, USA; Research Department, Shriners Hospitals for Children,
Cincinnati, OH, USA
Olivier Braissant • Laboratory of Biomechanics & Biocalorimetry, Coalition
for Clinical Morphology & Biomedical Engineering, University of Basel,
Basel, Switzerland
Claudio Brancolini • Dipartimento di Scienze e Tecnologie Biomediche,
Università degli Studi di Udine, Udine, Italy
Adeline Cambon-Binder • Laboratoire de Bio-ingéniérie et Biomécanique
Ostéo-articulaires, UMR, CNRS, 7052, Paris, France
Samantha Chan • ARTORG, Center for Biomedical Engineering Research,
Institute for Surgical Technology and Biomechanics, University of Bern,
Bern, Switzerland
Ewa M. Czekanska • AO Research Institute, Davos Platz, Switzerland
Alma U. “Dan” Daniels • Laboratory of Biomechanics & Biocalorimetry,
Coalition for Clinical Morphology & Biomedical Engineering, University of Basel,
Basel, Switzerland
Zbigniew Darzynkiewicz • Brander Cancer Research Institute, New York
Medical College, Valhalla, NY, USA
Stéphane Dhalluin • Investigative Non-Clinical Safety, Non-Clinical Development,
UCB Pharma SA, Braine-l’Alleud, Belgium
Benjamin Gantenbein-Ritter • ARTORG, Center for Biomedical Engineering
Research, Institute for Surgical Technology and Biomechanics, University of Bern,
Bern, Switzerland
Helga H.J. Gerets • Investigative Non-Clinical Safety, Non-Clinical Development,
UCB Pharma SA, Braine-l’Alleud, Belgium
Sibylle Grad • AO Research Institute, Davos Platz, Switzerland
Svenja Illien-Jünger • AO Research Institute, Davos Platz, Switzerland;
Department of Orthopaedics, Mount Sinai Medical Centre, New York, USA
Katharina Jähn • School of Dentistry Department of Oral Biology University
Missouri, Kansas City, Missouri, USA
ix
