METHODS IN MOLECULAR BIOLOGY” r& : y ; OR Ps Fapopteds __..Methods and-Protd¢éols Edited by Qs igh J. M. Brad NOT FOR SALE al ae. && SS 3K HUMANA Press METHODS EN MOLECULAR BIOLOGY™ 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. 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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 © 2004 Humana Press Ine ‘99 Riverview Drive, Suite 208 Toray, New Jersey 07512 hhummanapeess.com ny form or hy any mens. eletinie. enecheice:. photovepying, mucrofi ning, recording, ov theres Pinliser, Metlnds it Molecular Bialegy™r¢ 9 cadomark of the swithaul wrielen permissive Toa Thins Press Tne AIT papers. comments, epaiions, cunelusions, or recommendations are those of the muthartst, end do nor aeoessurlly eeflezr te view of the pulllsacr “This publicnsion is printed on ucidetnee ppes. 3d ANGST 739,18-1954 {Adnerican Scarulan Insjtu.s} Penuaaence 1! Paper for Printed Libeacy Matevials Production Editor: Rohia B. Weisberg, Cave illustrticin: Feom Fig. 1 in Caapter 11. “Metands for Culturing Priznaey Sympathetic Nourens Caveer desig by Pasivia F. 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INY RT 6543.21 1-s9259-512 946 ISBN} Libeary of Congress Catuluging-ia-Publication Duta Apoptosis methods anu protacols edited by ugh J. M. Brady prem, ‘Methods in molecular biology, TSSN WiRe.374S = 282) Inchidss hibliognaphival velere ISBN 0-N9603-873-4 Galk, paper) and ine IDALM 1. DNA Dare. phiticiogy Lariny Manuals. 2. Apopinbe-pysiokgy Mnuels- Qi 468 AC4I 2064] 1 Brady yg) M-1L Series Meth ineole. dar bik wake ‘QHOT Ase 20us S11 %do22 Linanatory Tinton, NEP WONT 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