Table Of ContentMETHODS IN MOLECULAR BIOLOGY”
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METHODS
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John M, Walker, Sexss Forror
207. Protein Nanotechuslogy: Protaras,
274, Photosynthesis Research Proweols, edited by
Yorn, 2605
296, Cell Cycle Protocol ite by Tin Humphrey and
Gavin Brooks. 2605
285. Immuntcherieul Proaucly Fad taro oud
toy Robor Buia, 205
294, Cell Migeation: Devetyornesia! Mesh
Protacas. lit by BureLin Guan, 2008
298, Laser Caplure Mficrudiseectivn: Methich and
Proc ote hy diane f. Sarnay ond
Stenbume Curran, 2005
WL DNA Viruses: SMethatt and Prot
Paul 4. Lieberman, 2155
281, Molecular Toviculogy Protocols, city
sf
i, elle 99
273, Batelets and Mogakersoeytes, Voluae 2:
Perspectives nnd Fechrignes.euited by
‘tomato ¥ ifebins and Meiyn P. Makan
Sor, 200
272, Platelets and Megakaryoeytes, Volume t=
Flenctioner!Agsays, Ce! hy JOM
{Gibine at Mareen P. Musut-Soth, 2004
271. B Coll Protoeols. eited by 2a Gu and Koos
Rujewshy. 2004
20, Parasite Genamies Protocols edited hy Sora
eniMetvites 2004
269, Vaceina Virws and Posvirology: Methods wid
Prisecits dived by Siwort leone, 2004
268, Public Health Wiceabiolagy: Meru and
seatacots edt r a
‘TFhouthane Keohavong and Stephen G. Grav. 20S
299, Basie Cell Culture. Hund Eaton,
Gaety! D, Helaaion and indy Stier 08
289. Kpidermal Cells, Weshads ars appticorions,
edited by Kursad Tarksen. 205
288. Oligonucleotide Synthesis, sectors i Appi
caus, eid by Piet Menten, 208
287. Epigeneties Protocols, sed hy Toygve 0.
Toller! 208
Aina L, Ragu de Spencer. 2009
267, Recombinant Gene Expression: Reviews and
Protas, Second Eating, edited by Powina
Iathse.aed Argetia dokason, 38
266, Genamies, Proteomics, and Clinical
Bacteriology: Methods wad Reviews. edited by
Neil Wolford ated Alan fonases, 2004
265. RNA Interference, Editing, und
Modification: Morkts and Pragerats. ited
286, Transgenic Plans Hethos and Protons,
sail by Lenaleo Pf, 2064
Call Cycle Contre] and Dysregulation
runacols: yen, yedimDependene Kinases,
vrs! Other Factors, eiled by Antonia Grea
264, Peoteln Areays: Metivals ond Protooals,
cted iy Erte Peng, 204
262. Flow Cyuumetrs, Second haution. ede by
Teresa §. Massey ana Robert G. laiey, 2004
262, Genetic Recombinaclon Protocols, ite hy
284, Siynal Teansduetion Protocols Szcand taht,
eke by Mabert C. Dickson aod Midas B.
Mendenbil. 108
onugation Protocols, edited by €
Nlemever, 2008
inte
282. Apnptasis Metiads and Protacol, ited by
Hgie JM. Brady. 2094
Astivabon
‘Alan §. Watidmon, 2004
261. Protein-Protein Interactions: Mths wat
Applicanons, edite hy lies fi. 2004
260, Mobile Genetic Elements: Protas and
Goonne Appiteanings edited hy Wosfgann
Miler and Bienes Copy. 2004
289, Reeeptur Signal Vrunsduction Protoculs,
Second Eauion, edited bg Gar 5 0. Willers
and R.A Folin Cholies, 2094
Sheet A. Sh hitb 2
280, Checkpoint Controls und Canser, Volume 1
Reviews sind Moael Svaeves. edited by Atel.
Sehonthais 200
219. Nitvie Oxide Protocols, Secor Falition,editsd
Gene Eup Mesh
Frotorots, edited hy Rickard A. Shimbets, 200
rand Metabolism: 3eiods on
Peas, elite by Dent R. Sehuerbe 3, 2098
256. Bacterial Artifical Chr mosomes, Volume 2:
Hunrstonal Studies, sited hy Shasony Zao
278, Protein NMR Techniques. Second Edivon,
ged by A. Kiivina Dowsing, 2004
Trinyelootide Repeat Protocols, edited by
285. Hactertal Artifieal Chrnimosomes, Volume 1
Linvore Conateivn, Bhvaeal Mtaneing 0d
Secquers ing eited by Shaving Zhao aad
276, Cupillury Electrophoresis of Proteins and
Peptides cated Mork AC Sirege a?
Avinash E Lagu. 204
hemoinfarmatics, cited hy Juryeu Raat,
04
Marvin So dolabe, 295
wae
Faibrye vate Live donrging, Tansgenests
fed Conia, eahied hy Heide Notaeen. 2008
253. Germ Cell Pentocals, Volume H: Seto on?
Gueyte Anobis.eiltby Heide Sate, 2008
METHODS IN MOLECULAR BIOLOGY™
Apoptosis Methods
and Protocols
Edited by
Hugh J. M. Brady
Institute of Child Health,
University College London,
London, UK
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Preface
The most fundamental question facing euch and every cell within an organ-
ism is to survive or to die, Cell deuth is required for normal function; some
estimates suggest that as many as one million cells undergo cell death every
second in the adult human body, Almost all cells undergoing physiological, or
programmed, cell death. independent of cell type, manifest a stercotypic pat-
tern of morphological changes termed apoptosis. Typically, apoptotic cells dis-
play shrinkage, membrane blebbing, chromatin condensalion, and nuclear
fragmentation. The integrity of the cell membrane is not lost during apoptosis
and so avoids eliciting the inflammatory response that would have been caused
by the spillage of the cell's contents, This is quite in contrast to the loss of cell
contents typical of necrosis, ‘The caspases. the family of intracellular cysteine
phological changes. Caspases cleave various substrate proteins that act.on DNA
fragmentation, nuclear envelupe integrily, the cytoskeleton, and cell volume
regulation. Apoptotic cells are cleared in vive by the process of phagocytosis,
in which specific “phagocytes” move to the site of apoptosis. engull the dying
cells anc digest them.
Apoptosis has a central role in many physiological processes, for example, in
the immune sysiem, Autoreactive cells are deleted via upoptosis to prevent
autoimmunily. AL the end of an immune response, activated lymphocytes are
removed fo maintain homeostasis within the immune system. Ln addition, im-
mune surveillance involves. the.use_of apoptosis to-remoxe aberrant cells that
are damaged or harbor a mutation, When the biochemical pathways regulating
apoptosis within the cell are defective in any way, this can make 4 major con-
Lribution to the development of pathological states. For example, insufficient
apoptosis can contribute to cancer, autuimmunity, or the persistence of infec-
tion, Extensive apoptosis is associated with many diseases, such as heart fail-
ure, ischemic injury, and newrodegencration. Therefore. procedures for
delecting apoptosis, quantifying apoptosis, understanding Ihe biochenustry of
apoptosis, and identifying the genes and proteins that regulate und carry out
Apoptosis are crucial techniques in 21s\-century biomedical research.
Apoptosis Methods and Protocols introduces the principle of each technique
and takes the reader through the procedure in a step-wise, user-(riendly fa
ion. ‘The clear explanations and inelusion of tips learned from many yeurs ex-
' 2 each technique allow. " ce h Scaystull:
vi Prejace
carry out procedures as diverse ay immunohistochemistry, Mow cytometry
kinase activity assays, yeast two-hybrid screening, of the cloning of novel genes
—py dlifferemiat expression -ay related to apoptosis,
Hugh }. M. Brady
Contents
Preiace
Contributors...
1 Measurement of Apoptosis by DNA Fragmentation
Demetrius Matassov, Terri Kagan, Julie Leblanc,
Marianna Sikorska, and Zahra Zakeri
2 Measurement of Caspase Activity in Cells Undergoing Apoptosis
Sharad Kumar.
3 Flow Cytometry-Based Methods for Apoptosis Detection
in Lymphoid Cells
Owen Williams
Apoptosis of Target Cells in Killer Cell Assays:
Richard C. Duke..
3. Detecting and Quantifying Apoptosis in Tissue Sections
Vicki Save, Peter A, Hall, and Philip }. Coates
6 Immunoassay for Single-Standed DNA in Apoptotic Cells
7 Mcthods to Measure Membrane Potential and Permeability
Transition in Mitochondria During Apoptosis
Naoufal Zamzami and Guide Kroemer .
@ Measurement of Changes in Infracellular Calcium
During Apoptosis
David J. MeCunkey and Leta Nutt.
9 Measurement of Changes in Apoptosis and Cell Cycle
Regulatory Kinase Cdk2
Gabriel Gil-Gémez
Carme Caelles and Ménica Morales
11 Methods for Culturing Primary Sympathetic Neurons
and for Determining Neuronal Viability
jonathan Whitteld, Stephen J. Neanie, and Jonathan Ham.
12. Immunacytachemical Techniques for Studying Apoptosis
in Primary Sympathetic Keurens
Stephen J. Neame, Jonathan Whittield, and Jonathan HaM vm. 169
vill
Contents
Measuring Programmed Cell Dicath in Plants
Ludmila Rizhsky, Vladimir Shulacv, and Ron Mittler. 179
16
V7
Detection of apoptosis tr Prosaphitar
Kimberly McCall and Jeanne S, Peterson ....
Measurement of Apoptotic Cell Clearance In Vitro
Andrew Devitt and Christopher D, Gregory
Yeast fwo-Hybrid Screening for Proteins Interacung,
With the Anti-Apoptotic Protein A20
Karen Heyninck, Sofie Van Huffel, Marja Keeike,
and Rudi Beyaert
The Yeast Three-Hybrid System As a Too! to Study Caspases
Wim van Criekinge, Peter Schatte, Karen Heyninck,
191
207
223
18
19
and Rudi Beyaert
Cloning of Apoptosis Related Genes by Representational
Dilference Analysis of CDNA
Michael Hubank, Fredrik Bryntesson, jennifer Regan,
and David G. SHAE sesso
Identification of Apoptosis Regulalory Genes Using
Jnsertional Mutagenesi
Index
Joéle Thomas, Yann Levercier, Anne-Laure Mathieu,
and jacqueline Marvel 275
297
Contributors
Rupr Beyacer + Department of Molecular Biomedical Research, Flanders
Interuniversity Institute for Biotechnology, Ghent University, Ghent,
Belgium
Huss JM. Beapy + Molecular Hematology and Cancer Biology Unit,
Institute of Child Health, University College London. London, UK
FREDRIK BeYNiRsson + Molecular Hematology and Cancer Biology Uuit.
Institute of Child Health, University College London, London, UR
Cann CaELLES * Institut de Recerca Biomédicu de Barcelona-Pare Cientific
de Rarcetona, Department of Biochemistry and Molecular Biology.
University of Barcelona, Barcelona, Spain
PuiLir I. Coxes + Division of Pathology and Neurosciences, Ninewells Hospital
Medticats University of Dundee, Dundee-Scottend:
Anpeew Devitt » Centre for Inflammation Research, The University
of Edinvurgh Medical School. Edinburgh, UK
Rienarp C. Duke » CERES Pharmaceuticals Ltd., Medical Oncology.
University of Colorado Health Science Center, and Eleanor Roosevelt
Institute for Cancer Research, Denver, CO
Oskar S. Pranxruer + University of Miami Medical School and Apostain
Inc., Miami, FA.
Gaprirt Gu-Gomez « Institut Municipal d'Iuvestigacié Médica i Universitat
Popes Fabra, Barcelona, Spain
Cuerstopner D. Grecory + ¢ tre Inflammation Rese The
University of Edinburgh Medical School, Edinburgh, UK
Perce A. Hai + Department of Pathology and Cancer Research Centre,
Queen's University, Belfast. Northern Ireland. UK
Jonathan Ham » Molecular Hematology and Cancer Biology Unit-Anstitute
of Child Health, University College Landon, London, UR
Kaen Heysinek * Department of Molecular Biomedical Research, Flanders
Titeruniver sity Institute for Biotechnology, Chent University, Ghent,
Belgium
Micnatt, Houank * Molecular Hematology and Cancer Biology Unit,
Institute of Child Health, University College Landon, London, UK
Terri Kagan + Department of Biology, Queens College and Graduate
Center, City University of New York, Flushing. NY
Maasa Kreme» De; Molecular B: R fi, Flande|
Interuniversity Lustitute for Biotechnology, Ghent University, Ghent,
Belgium
ix
x Contributors
Guino Keormer » CNRS-UMR 8/25, institut Gustave Roussy, Villejuif.
France
Suanau Kusiar + Hanson instinete fnstiture of ttedicat and Veterinary
Science and Department of Medicine, University of Adelaide, Adelaide,
Australia
Iuut Leatanc + frstiture for Biological Sciences. National Royal Council,
Oitawa, Ontario, Canada
Yau Leverrier + Laboratory of Molecular and Cellular Biology.
Recherche CNRSIENS, Lyon, France
Jacquetine Maven, * Laboratory of Molecular and Cellular Biology, Unité
de Recherche CNRSIENS, Lyon, France
Demerauis Marassoy + Department of Biology, Queens College
G ne Oi City University of New Yark, FI if NY.
inité de
ANSE-LAURE MATHIEU + Laboratory of Molecular and Cellular Biology.
Unité de Recherche CNRSIENS, Lyon, France
Kinmerty McCatt » Department of Biology, Boston University, Boston, MA
Davin J- MeCoviiy-« Department of Cancer Biology, The University af
Texas M.D. Anderson Cancer Center, Houston, TX
Ron Mirrien + Department of Biochemistry, University of Nevada, Reno NV
Moxica Moraes Division of Molecular Biology. Memorial Sloan-
Kettering Cancer Center, New York, NY
Steenen J. NeAME + £isai London Research Laboratories, University
College London, London, UK
Leta NUTT + Department of Cancer Biology, The University of Texas MD.
Anderson Cancer Center, Houston, TX
Jeanne 8. Perenson * Department of Biology, Boston University, Boston, MA
Jexniene Recan * Department af Anatomy and Developmental Biology.
University College, London, UK
Lupsita Rizasky * Department of Botany, Plant Sciences Institute, lowa
tiniversite; Ames, tt
Vick Savi + Department of Pathology, Addenbrovkes Hospital, Cambridge
University, Cambridge, UK
Daviv G. Scuarz« Section of hmmunobiology, Howard Hughes Medical
Instinute, Yate University. New Haven. CT
Perak Scorn: +* Deparoment of Molecular Biomedical Research, Flanders
imeruniversity Institute for Biotechnology, Ghent University, Ghent,
Belgium
VLADIMIR SHCLAEV * Virginia Bioinformatics Institute, Blacksburg, VA
Mariasna Sikorska + /nstitute for Biological Sciences, National Royal
Council, Ottawa, Ontario, Canada
