Flow Cytometry. Principles and Applications. Edited by. Marion G. Macey, PhD

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1 Flow Cytometry

2 Flow Cytometry Principles and Applications Edited by Marion G. Macey, PhD Department of Haematology The Royal London Hospital London, United Kingdom

3 2007 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. All papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the publisher. This publication is printed on acid-free paper. ANSI Z (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover design by Nancy K. Fallatt Cover illustration:(from left) typical lens and mirror assembly for detection of 90 LS, FITC, PE, and ECD fluorescence (above; Fig. 8, Chap. 1, see full caption on p. 8); flow cytometric enumeration of cells that have migrated through wells containing HUVECs (below; Fig. 3, Chap. 10, see complete figure and caption on p. 230); fluorescence emission spectra for FITC and PE (Fig. 6, Chap. 1, see complete caption on p. 12); chemical structure of a probe for estimating intracellular oxidants (Fig. 10B, Chap. 3, see complete figure and caption on pp ); the light-scattering properties of platelets in diluted whole blood (Fig. 6, Chap. 10, see discussion on p. 244). For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: ; Fax: ; orders@humanapr.com; or visit our Website: Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $30.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arrangedand is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [ /07 $30.00]. Printed in the United States of America eisbn 10-digit: eisbn 13-digit: Library of Congress Cataloging-in-Publication Data Flow cytometry : principles and applications / edited by Marion G. Macey. p. ; cm. Includes bibliographical references and index. ISBN 10-digit: (alk. paper) ISBN 13-digit: (alk. paper) 1. Flow cytometry. 2. Cell separation. 3. Cytodiagnosis. I. Macey, Marion G. [DNLM: 1. Flow Cytometry--methods. 2. Cell Separation--methods. 3. Cytodiagnosis--methods. QY 95 F ] QH585.5.F56F '582--dc

4 Preface Improvements in instrument design and computing power and the increased availability of fluorescent agents have led to an increased usage of flow cytometry in both the research and clinical settings. Flow Cytometry: Principles and Applications provides a comprehensive introduction to data interpretation, quality control procedures, pitfalls and problems, in addition to detailed protocols from leading authorities with extensive practical experience in flow cytometry. Flow Cytometry: Principles and Applications also presents the principles and potential of flow cytometry to assess many functional aspects of cells. This is an essential handbook and reference to both new and experienced flow cytometry users. Marion G. Macey, PhD Consultant Clinical Scientist Professor of Immunohaematology Department of Haematology The Royal London Hospital London, United Kingdom v

5 Contents Preface...v Contributors...ix 1 Principles of Flow Cytometry Marion G. Macey Cell Preparation Desmond A. McCarthy Fluorochromes and Fluorescence Desmond A. McCarthy Quality Control in Flow Cytometry David Barnett and John T. Reilly Experimental Design, Data Analysis, and Fluorescence Quantitation Mark W. Lowdell Apoptosis Detection by Flow Cytometry Paul Allen and Derek Davies DNA Analysis by Flow Cytometry Derek Davies and Paul Allen Immunological Studies of Human Cells Ulrika Johansson Calcium: Cytoplasmic, Mitochondrial, Endoplasmic Reticulum, and Flux Measurements Gary Warnes and Marion G. Macey Further Functional Studies Marion G. Macey Cell Sorting by Flow Cytometry Derek Davies Appendix: Useful Internet Sites and Suppliers Index vii

6 Contributors PAUL ALLEN, BSc, PhD Centre for Haematology, Institute of Cell and Molecular Sciences, Barts and The London School of Medicine and Dentistry, London, United Kingdom DAVID BARNETT, PhD, MRCPath UKNEQAS, Sheffield, United Kingdom DEREK DAVIES, BSc FACS Laboratory, London Research Institute, Cancer Research UK, London, United Kingdom ULRIKA JOHANSSON, BSc, PhD Department of Haematology, The Institute of Cell and Molecular Science, Queen Mary University London, London, United Kingdom MARK W. LOWDELL, MSc, PhD, FRCPath Senior Lecturer in Haematology, Director of Laboratory of Cellular Therapeutics, The Royal Free and UCL Medical School, London, United Kingdom MARION G. MACEY, BSc, PhD, FRCPath Professor, Department of Haematology, The Royal London Hospital, London, United Kingdom DESMOND MCCARTHY, BSc, PhD School of Biological Sciences, Queen Mary University London, London, United Kingdom JOHN T. REILLY, MD, FRCPath Professor, UKNEQAS, Sheffield, United Kingdom GARY WARNES, BSc, PhD Imaging and Flow Cytometry Facility, The Institute of Cell and Molecular Sciences, Queen Mary University London, London, United Kingdom ix

Flow Cytometry. Principles and Applications. Edited by. Marion G. Macey, PhD

Flow Cytometry. Principles and Applications. Edited by. Marion G. Macey, PhD Flow Cytometry Flow Cytometry Principles and Applications Edited by Marion G. Macey, PhD Department of Haematology The Royal London Hospital London, United Kingdom 2007 Humana Press Inc. 999 Riverview