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STUDZINSKI Department of Pathology and Laboratory Medicine UMD—New Jersey Medical School Newark, N.J., USA OXFORD UNIVERSITY PRESS OXFORD UNIVERSITY PRESS Great Clarendon Street, Oxford OX2 6DP Oxford University Press is a department of the University of Oxford and furthers the University's aim of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Athens Auckland Bangkok Bogota Buenos Aires Calcutta Cape Town Chennai Dar es Salaam Delhi Florence Hong Kong Istanbul Karachi Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Paris Sao Paulo Singapore Taipei Tokyo Toronto Warsaw and associated companies in Berlin Ibadan Oxford is a registered trade mark of Oxford University Press Published in the United States by Oxford University Press Inc., New York © Oxford University Press, 1999 All rights reserved. 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A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data (Data available) ISBN 0-19-963784-9 (Hbk) 0-19-963785-7 (Pbk) Typeset by Footnote Graphics, Warminster, Wilts Printed in Great Britain by Information Press, Ltd, Eynsham, Oxon. Preface Apoptosis, a concept derived from pathological observations dating back to the availability of the microscope, has been recently afforded treatment reminiscent of the ancient apocalyptic writings. Revelations are reported daily of new, and so it is claimed, profound insights into cellular survival mecha- nisms, and their principal default pathway, apoptosis. The preoccupation of many scientists with this cellular programme, or programmes, appears to be driven by several currents. There is a bewildering array of commercial reagents and accompanying literature reporting to provide, often quick and easy, means of discovering important secrets of nature. There is also the fascination of the scientists with the beauty of an almost endless cascades of protein-protein interactions that lead to an irrevocable end-point—cell death. And of course, there is the legitimate expectation that important components of therapy for cancer and immune diseases will be based on our understanding of the precise mechanisms of these apoptotic cascades. This volume presents the techniques essential for contemporary research on diverse aspects of apoptosis. In addition to the basic methodology for recogni- tion of the apoptotic phenotype and its characteristic DNA fragmentation, the text contains a wide variety of procedures used to investigate the mechanistic aspects of the programmes for survival or death of mammalian cells. A team of scientists who are among the leaders in apoptosis research has provided numerous protocols which describe in detail how to perform these procedures and discusses them from the individual points of view of each contributor. The protocols most frequently used in current investigations of apopotosis research are presented with variations that have been found particularly useful for a par- ticular application, thus allowing the reader to benefit from the experience of laboratories which focus on different aspects of apoptosis research. Attention is also directed to the choice of the procedures, to pitfalls in their execution, and to critical interpretation of the results. It is believed that the nuances of technical approaches discussed here will be helpful to the experi- enced as well as the beginning investigators. The credit for this must go to the team of authors and the OUP staff. New Jersey G.P.S. 1999 This page intentionally left blank Contents List of Contributors Abbreviations 1. Overview of apoptosis George P. Studzinski 1. General introduction and overview of contents 2. Historical perspective 3. Distinction of apoptosis from other forms of cell death 4. Apoptotic cascades 5. Time course of apoptotic cascades 6. Selection of methods Procedure for determination of increased mitochondrial to nuclear DNA ratio, for the detection and quantitation of apoptosis 7. Pitfalls References 2. Morphological recognition of apoptotic cells James W. Wilson and Christopher S. Potten 1. Introduction Key morphological features of apoptotic cells 2. Light and fluorescent microscopy techniques for the assessment of apoptosis Preparation of cell or tissue samples Nuclear counterstains 3. Electron microscopic techniques 4. Quantitation of apoptotic events Methods Problems in scoring apoptotic events 5. In situ detection of DNA strand breaks 6. Other techniques 7. Conclusions References xv xix 1 1 5 5 8 8 11 12 14 16 19 19 19 20 20 23 27 29 29 31 33 37 38 38