Table Of ContentM M B ™
ETHODS IN OLECULAR IOLOGY
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John M. Walker
School of Life Sciences
University of Hertfordshire
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Apoptosis
Methods and Protocols
Second Edition
Edited by
Peter Erhardt and Ambrus Toth
Boston Biomedical Research Institute, Watertown, MA, USA
Editors
Peter Erhardt Ambrus Toth
Boston Biomedical Research Institute Boston Biomedical Research Institute
Watertown, MA Watertown, MA
USA USA
ISSN 1064-3745 e-ISSN 1940-6029
ISBN 978-1-60327-016-8 e-ISBN 978-1-60327-017-5
DOI 10.1007/978-1-60327-017-5
Springer Dordrecht Heidelberg London New York
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Cover illustration: Apoptotic cells display the same distinct morphology detectable by DIC microscopy in different
embryonic development stages (Fig. 25-2).
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Preface
The ability to detect and quantify apoptosis, to understand its biochemistry, and to iden-
tify its regulatory genes and proteins is crucial to biomedical research. In this second
edition of Apoptosis in Methods in Molecular Biology, expert researchers describe the tech-
niques to best investigate the critical steps involved in the apoptotic process.
These readily reproducible step-by-step instructions are presented from several
different research perspectives. The first part of the book provides an overview on the
general techniques to detect apoptotic cell death, including methods such as caspase
activity measurement, flow cytometry, live cell imaging, histopathology, and apoptosis
detection in cell-free systems. In contrast, the second part lists methods to assess two
forms of non-apoptotic cell death, necroptosis and autophagy.
Apoptotic proteins often undergo posttranslational modifications that alter their activ-
ity toward their downstream substrates. Techniques are described to analyze transglutam-
ination, S-nitrosylation, and redox modifications of apoptotic proteins. Subsequently,
several chapters are devoted to techniques that help dissect the major regulatory pathways
of cell death and survival, including p53-dependent and independent and cell cycle regu-
latory proteins; the role of mitochondrial membrane permeabilization, unfolded protein
response and ER stress, uncoupling protein-2, and microRNAs in programmed cell death;
as well as the mechanism of phagocytosis by macrophages.
The fifth part of the book contains specific methodology required to evaluate
apoptosis in various organs such as central nervous and cardiovascular system, myeloid
progenitor cells as well as skeletal muscle. Techniques to detect apoptotic cell death d uring
mammalian development are also described here. The final part of the book summarizes
the approaches to study apoptosis in nonmammalian model organisms such as yeast,
Drosophila, and Caenorhabditis elegans.
The protocols follow the Methods in Molecular Biology series format, each of them
offering detailed laboratory instructions, an introduction outlining the principle behind
the technique, lists of equipment and reagents, and tips on troubleshooting on how to
avoid common pitfalls.
Apoptosis: Methods and Protocols, Second Edition, constitutes a key technical reference
to the significant methodologies used in the field, and offers beginners and experienced
researchers powerful tools to illuminate the phenomena of programmed cell death.
Watertown, MA Peter Erhardt
August 2008 Ambrus Toth
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I: DETECTION OF APOPTOSIS
1 Analysing Caspase Activation and Caspase Activity in Apoptotic Cells . . . . . . . . . . 3
Sharad Kumar and Loretta Dorstyn
2 Flow Cytometry-Based Apoptosis Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Donald Wlodkowic, Joanna Skommer, and Zbigniew Darzynkiewicz
3 Live to Dead Cell Imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Stephen W.G. Tait, Lisa Bouchier-Hayes, Andrew Oberst,
Samuel Connell, and Douglas R. Green
4 Detection of Apoptosis in Tissue Sections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Eva Csizmadia and Vilmos Csizmadia
5 Detection of Apoptosis in Cell-Free Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Dhyan Chandra and Dean G. Tang
PART II: DETECTION OF NON-APOPTOTIC CELL DEATH
6 Methods to Analyze Cellular Necroptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Benchun Miao and Alexei Degterev
7 Detection of Cell Death by Autophagy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Narasimman Gurusamy and Dipak K. Das
PART III: MODIFICATIONS OF APOPTOTIC PROTEINS DURING APOPTOSIS
8 Methods to Analyze Transglutamination of Proteins Involved in Apoptosis. . . . . . 107
Zoltán Nemes and László Fésüs
9 Methods to Analyze S-nitrosylation of Proteins Involved in Apoptosis. . . . . . . . . . 117
Neelam Azad, Anand Krishnan V. Iyer, and Yon Rojanasakul
10 Application of In Vivo EPR for Tissue pO and Redox Measurements . . . . . . . . . 131
2
Nadeem Khan and Dipak K. Das
PART IV: ANALYSIS OF THE FUNCTION OF MAJOR REGULATORS
OF APOPTOSIS/CELL SURVIVAL
11 Assays to Measure p53-Dependent and -Independent Apoptosis. . . . . . . . . . . . . . 143
Darren C. Phillips, Sean P. Garrison, John R. Jeffers,
and Gerard P. Zambetti
12 Measurement of Changes in Cdk2 and Cyclin O-Associated
Kinase Activity in Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Ramon Roset and Gabriel Gil-Gómez
vii
viii Contents
13 Fluorometric Methods for Detection of Mitochondrial
Membrane Permeabilization in Apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Soumya Sinha Roy and György Hajnóczky
14 Regulation of Apoptosis by the Unfolded Protein Response . . . . . . . . . . . . . . . . . 191
Andrew Fribley, Kezhong Zhang, and Randal J. Kaufman
15 Detection of Uncoupling Protein-2 (UCP2)
As a Mitochondrial Modulator of Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Zoltan Derdak, Tamako A. Garcia, and Gyorgy Baffy
16 Multiple Approach to Analyzing the Role of MicroRNAs in Apoptosis . . . . . . . . . 219
Riccardo Spizzo and George A. Calin
17 Assessment of Apoptotic Cell Phagocytosis by Macrophages. . . . . . . . . . . . . . . . . 247
Kathleen A. McPhillips and Lars-Peter Erwig
PART V: ANALYSIS OF APOPTOSIS IN DIFFERENT ORGANS
18 Detection of Apoptosis in Mammalian Development . . . . . . . . . . . . . . . . . . . . . . 259
Lin Lin, Carlos Penaloza, Yixia Ye, Richard A. Lockshin,
and Zahra Zakeri
19 Detection of Apoptosis in the Central Nervous System. . . . . . . . . . . . . . . . . . . . . 273
Youngsoo Lee and Peter J. McKinnon
20 Genetic Mapping of Anti-Apoptosis Pathways in Myeloid Progenitor Cells. . . . . . 283
Dan Liu and Zhou Songyang
21 Analysis of Apoptosis in Isolated Primary Cardiac Myocytes . . . . . . . . . . . . . . . . . 293
Adel Mandl, Ambrus Toth, and Peter Erhardt
22 Cell Death in Myoblasts and Muscles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Lawrence M. Schwartz, Zhengliang Gao, Christine Brown,
Sangram S. Parelkar, and Honor Glenn
PART VI: ANALYSIS OF APOPTOSIS IN MODEL ORGANISMS
23 Reliable Method for Detection of Programmed Cell Death in Yeast . . . . . . . . . . . 335
Xinchen Teng and J. Marie Hardwick
24 Detection of Cell Death in Drosophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Kimberly McCall, Jeanne S. Peterson, and Tracy L. Pritchett
25 Detecting Apoptotic Cells and Monitoring Their Clearance
in the Nematode Caenorhabditis elegans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
Nan Lu, Xiaomeng Yu, Xiangwei He, and Zheng Zhou
26 Detection of Herpes Simplex Virus Dependent Apoptosis. . . . . . . . . . . . . . . . . . . 371
Christopher R. Cotter and John A. Blaho
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Contributors
NEELAM AZAD (cid:129) Department of Pharmaceutical Sciences, West Virginia University,
Morgantown, WV, USA
GYORGY BAFFY (cid:129) Division of Gastroenterology & Liver Research Center, Brown
Medical School and Rhode Island Hospital, Providence, RI, USA; Brigham and
Women’s Hospital, Harvard Medical School and VA Boston Healthcare System,
Boston, MA, USA
JOHN A. BLAHO (cid:129) Department of Microbiology, Mount Sinai School of Medicine,
New York, NY, USA
LISA BOUCHIER-HAYES (cid:129) Department of Immunology, St. Jude Children’s Research
Hospital, Memphis, TN, USA
CHRISTINE BROWN (cid:129) Biology Department, University of Massachusetts, Amherst,
MA, USA
GEORGE A. CALIN (cid:129) Department of Experimental Therapeutics, The University of
Texas M.D. Anderson Cancer Center, Houston, TX, USA
DHYAN CHANDRA (cid:129) Department of Pharmacology and Therapeutics, Roswell Park
Cancer Institute, Buffalo, NY, USA
SAMUEL CONNELL (cid:129) Department of Immunology, St. Jude Children’s Research
Hospital, Memphis, TN, USA
CHRISTOPHER R. COTTER (cid:129) Department of Microbiology, Mount Sinai School of
Medicine, New York, NY, USA
EVA CSIZMADIA (cid:129) Center for Vascular Biology, Beth Israel Deaconess Medical Center,
Boston, MA, USA
VILMOS CSIZMADIA (cid:129) Millennium Pharmaceuticals, Inc., Cambridge, MA, USA
ZBIGNIEW DARZYNKIEWICZ (cid:129) Brander Cancer Research Institute, New York Medical
College, Valhalla, NY, USA
DIPAK K. DAS (cid:129) Cardiovascular Research Center, University of Connecticut,
School of Medicine, Farmington, CT, USA
ALEXEI DEGTEREV (cid:129) Department of Biochemistry, Tufts University School of Medicine,
Boston, MA, USA
ZOLTAN DERDAK (cid:129) Division of Gastroenterology & Liver Research Center,
Brown Medical School and Rhode Island Hospital, Providence, RI, USA
LORETTA DORSTYN (cid:129) Hanson Institute, Adelaide, Australia
PETER ERHARDT (cid:129) Boston Biomedical Research Institute, Watertown, MA, USA
LARS-PETER ERWIG (cid:129) Department of Medicine and Therapeutics, Institute of Medical
Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK
LASZLO FESUS (cid:129) Departments of Biochemistry and Molecular Biology and Signaling
and Apoptosis Research Group, Hungarian Academy of Sciences, Research Center
for Molecular Medicine, University of Debrecen Medical and Health Sciences
Center, Debrecen, Hungary
ix
x Contributors
ANDREW FRIBLEY (cid:129) Department of Biological Chemistry, The University of Michigan
Medical Center, Ann Arbor, MI, USA
ZHENGLIANG GAO (cid:129) Molecular and Cellular Biology Program, University of
Massachusetts, Amherst, MA, USA
TAMAKO A. GARCIA (cid:129) Division of Gastroenterology & Liver Research Center, Brown
Medical School and Rhode Island Hospital, Providence, RI, USA
SEAN P. GARRISON (cid:129) Department of Biochemistry, St. Jude Children’s Research
Hospital, Memphis, TN, USA
GABRIEL GIL-GOMEZ (cid:129) Institut Municipal d’Investigació Mèdica (IMIM),
Barcelona, Spain
HONOR GLENN (cid:129) Pioneer Valley Life Sciences Institute, Springfield, MA, USA
DOUGLAS R. GREEN (cid:129) Department of Immunology, St. Jude Children’s Research
Hospital, Memphis, TN, USA
NARASIMMAN GURUSAMY (cid:129) Cardiovascular Research Center, University of
Connecticut, School of Medicine, Farmington, CT, USA
GYÖRGY HAJNÓCZKY (cid:129) Department of Pathology, Anatomy and Cell Biology,
Thomas Jefferson University, Philadelphia, PA, USA
J. MARIE HARDWICK (cid:129) Department of Pharmacology and Molecular Sciences,
Johns Hopkins School of Medicine, Baltimore, MD, USA; W. Harry Feinstone
Department of Molecular Microbiology and Immunology, Johns Hopkins University
Bloomberg School and Public Health, Baltimore, MD, USA
XIANGWEI HE (cid:129) Department of Human and Molecular Genetics, Baylor College of
Medicine, Houston, TX, USA
ANAND KRISHNAN V. IYER (cid:129) Department of Pharmaceutical Sciences, West Virginia
University, Morgantown, WV, USA
JOHN R. JEFFERS (cid:129) Department of Biochemistry, St. Jude Children’s Research Hospital,
Memphis, TN, USA
RANDAL J. KAUFMAN (cid:129) Departments of Biological Chemistry, Internal Medicine and
the Howard Hughes Medical Institute, The University of Michigan Medical Center,
Ann Arbor, MI, USA
NADEEM KHAN (cid:129) EPR Center for Viable Systems, Dartmouth Medical School,
Hanover, NH, USA
SHARAD KUMAR (cid:129) Hanson Institute, Adelaide, Australia
YOUNGSOO LEE (cid:129) Department of Genetics and Tumor Cell Biology, St. Jude Children’s
Research Hospital, Memphis, TN, USA
LIN LIN (cid:129) Medarex Inc., Bloomsbury, NY, USA
DAN LIU (cid:129) Verna and Marrs McLean Department of Biochemistry and Molecular
Biology, Baylor College of Medicine, Houston, TX, USA
RICHARD A. LOCKSHIN (cid:129) Department of Biological Sciences, St. John’s University,
Queens, NY, USA
NAN LU (cid:129) Verna and Marrs McLean Department of Biochemistry and Molecular
Biology, Baylor College of Medicine, Houston, TX, USA
ADEL MANDL (cid:129) Boston Biomedical Research Institute, Watertown, MA, USA
KIMBERLY MCCALL (cid:129) Department of Biology, Boston University, Boston, MA, USA
PETER J. MCKINNON (cid:129) Department of Genetics and Tumor Cell Biology,
St. Jude Children’s Research Hospital, Memphis, TN, USA
KATHLEEN A. MCPHILLIPS (cid:129) Department of Pediatrics, National Jewish Medical and
Research Center, Denver, CO, USA
Contributors xi
BENCHUN MIAO (cid:129) Department of Biochemistry, Tufts University School of Medicine,
Boston, MA, USA
ZOLTÁN NEMES (cid:129) Departments of Psychiatry and Signaling and Apoptosis Research
Group, Hungarian Academy of Sciences, Research Center for Molecular Medicine,
University of Debrecen Medical and Health Sciences Center, Debrecen, Hungary
ANDREW OBERST (cid:129) Department of Immunology, St. Jude Children’s Research Hospital,
Memphis, TN, USA
SANGRAM S. PARELKAR (cid:129) Molecular and Cellular Biology Program, University of
Massachusetts, Amherst, MA, USA
CARLOS PENALOZA (cid:129) Department of Biology, Queens College, Flushing, NY, USA
JEANNE S. PETERSON (cid:129) Department of Biology, Boston University, Boston, MA, USA
DARREN C. PHILLIPS (cid:129) Department of Biochemistry, St. Jude Children’s Research
Hospital, Memphis, TN, USA
TRACY L. PRITCHETT (cid:129) Department of Biology, Boston University, Boston, MA, USA
YON ROJANASAKUL (cid:129) Department of Pharmaceutical Sciences, West Virginia
University, Morgantown, WV, USA
RAMON ROSET (cid:129) Institut Municipal d’Investigació Mèdica (IMIM), Barcelona, Spain
SOUMYA SINHA ROY (cid:129) Department of Pathology, Anatomy and Cell Biology, Thomas
Jefferson University, Philadelphia, PA, USA
LAWRENCE M. SCHWARTZ (cid:129) Pioneer Valley Life Sciences Institute, Springfield,
MA, USA; Biology Department, University of Massachusetts, Amherst, MA, USA
JOANNA SKOMMER (cid:129) Queen’s Medical Research Institute, Edinburgh, UK
ZHOU SONGYANG (cid:129) Verna and Marrs McLean Department of Biochemistry and
Molecular Biology, Baylor College of Medicine, Houston, TX, USA
RICCARDO SPIZZO (cid:129) Department of Experimental Therapeutics, The University of
Texas M.D. Anderson Cancer Center, Houston, TX, USA
STEPHEN W.G. TAIT (cid:129) Department of Immunology, St. Jude Children’s Research
Hospital, Memphis, TN, USA
DEAN G. TANG (cid:129) Department of Carcinogenesis, The University of Texas M.D.
Anderson Cancer Center, Smithville, TX, USA
XINCHEN TENG (cid:129) Department of Pharmacology and Molecular Sciences, Johns Hopkins
School of Medicine, Baltimore, MD, USA
AMBRUS TOTH (cid:129) Charles River Laboratories, Wilmington, MA, USA
DONALD WLODKOWIC (cid:129) The Bioelectronics Research Center, University of Glasgow,
Glasgow, UK
YIXIA YE (cid:129) Department of Biology, Queens College, Flushing, NY, USA
XIAOMENG YU (cid:129) Verna and Marrs McLean Department of Biochemistry and
Molecular Biology, Baylor College of Medicine, Houston, TX, USA
ZAHRA ZAKERI (cid:129) Department of Biology, Queens College, Flushing, NY, USA
GERARD P. ZAMBETTI (cid:129) Department of Biochemistry, St. Jude Children’s Research
Hospital, Memphis, TN, USA
KEZHONG ZHANG (cid:129) Department of Biological Chemistry, The University of Michigan
Medical Center, Ann Arbor, MI, USA; Center for Molecular Medicine and
Genetics, Wayne State University School of Medicine, Detroit, MI, USA
ZHENG ZHOU (cid:129) Verna and Marrs McLean Department of Biochemistry and
Molecular Biology, Baylor College of Medicine, Houston, TX, USA
Part I
Detection of Apoptosis
