METHODS IN CELL BIOLOGY Prepared under the Auspices of the American Society for Cell Biology VOLUME 33 Flow Cytometry Edited by ZBIGNIEW DARZYNKIEWICZ CANCER RESEARCH INSTITUTE OF THE NEW YORK MEDICAL COLLEGE AT VALHALLA VALHALLA, NEW YORK 10595 HARRY A. CRISSMAN BIOMEDICAL SCIENCES DIVISION LQS ALAMOS NATIONAL LABORATORY LOS ALAMOS, NEW MEXICO 81545 ACADEMIC PRESS, INC. Harcourl Brace Jovanovich, Publishers San Diego New York Boston London Sydney Tokyo Toronto Series Editor LESLIE WILSON Department of Biological Sciences University of California Santa Barbam, California This book is printed on acid-free paper. @ Copyright 0 1990 by Academic Press, Inc. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. San Diego, California 92101 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NWl 7DX Library of Congress Catalog Card Number: 64-14220 ISBN 0-12-564133-8 (alk. paper) ISBN 0-12-203050-8 (pbk. : alk. paper) Printed in the United States of America 9 0 9 1 9 2 9 3 9 8 7 6 5 4 3 2 1 Numbers in parentheses indicate the pages on which the authors' contributions begin. Hemz BALSCH, Institut fiir Biophysik und Strah- DAVIDJ . CHAPLIN, Medical Biophysics Unit, enbiologie, Universitat Hamburg, D-2000 B. C. Cancer Research Centre, Vancouver, Hamburg 20, Federal Republic of Germany British Columbia, V5Z lL3, Canada (509) (217) YUHCHYAUC ZEN, Department of Pathology, Mmw E Bmmom~L, ife Sciences Division. Los School of medic in^ University of B n , Alamos National Laboratory, University of Seattle, Washington 98195 (185) California, Los Alamos, New Mexico 87545 (369) In JISRLCEH IUSTENSTEhNe ,F insen Laboratory, Rigshospitalet, University Hospital, DK-2100 KEmm D. BAUlin, Department of Pathology, Copenhagen, Denmark (127) Northwestern University School of Medicine, Chicago, Illinois 60611 (235) GAETANOC IANCIOD, epartment of Surgery, Jackson Memorial Hospital, Miami, Florida WOLEGANGB EIXERB, iomedical Sciences Divi- 33101 (19) sion, Lawrence Livermore National Labora- tory, Livermore, California 94550 (207) L. ScmC RAM, Life Sciences Division, Los Ala- mos National Laboratory, University of Cal- CATHERINBEE RGOUNIOULXa,b oratoire de ifornia, Los Alamos, New MBLim 87545 (369. Physiologie VkgWe Molkulaire, Facultk des 377) Sciences, Universitk d'Orsay, 91405 Orsay, France (563) HARRYA. CRISSMANCe. ll Biology Group, Los Alamos National Laboratory, Los Alamos. R.UPH M. B~~HMLEuRdw, ig Institute for Cancer New Mexico 87545 (89,97,1 05, 199,305) Research, Melbourne 'Hunour Biology Bmch, Melbourne, Victoria 3050, Australia (173) JOHN D. Crussw, Department of Pathology, Wayne State University, Detroit, Michigan E Rh~ ,Dep artment of Biophysics, Institute 48202 (1) for Cancer Research, Montebello, 0310 Oslo, Norway (519) ZBIGNIEWDA RZYNKIEWICCZa,n cer Research SPENCERC . BROWNC, ytomktrie, Institut des Institute of the New York Medical College at Sciences Vkgktales, Centre National de la Valhalla, Valhalla, New York 10595 (285,305, Recherche Scientifique, 91198 Gif-sur-Yvette, 337, 655) France (563) LARRLY. DEAVENL. ife Sciences Division, Los M,uty CAMPBELLLif, e Sciences Division. Los Alamos National Laboratory, University of Alamos National Laboratory, University of California, Los Alamos, New Mexico 87545 California, Los Alamos, New Mexico 87545 (377) (377) FRANK DOLBEARBEi,o medical Sciences Divi- ROBERTC ERRAD, epartment of Surgery, Henry sion, Lawrence Livermore National Labora- Ford Hospital, Detroit, Michigan 48202 (1) tory, Livermore, California 94550 (81.207) xi xii CONTRIBUTORS LYNNG .D RESSLERD, ivision of Molecular and ANGELIKAG ROSSMA", Department of Com- Cellular Diagnostics, University of New Mex- parative Medicine, School of Medicine, ico Cancer Center, University of New Mex- University of Washington, Seattle, ico School of Medicine, Albuquerque, New Washington 98195 (185) Mexico 87131 (157) DAMDW . HEDLEYD, epartments of Medicine RALPHE . DURANDM, edical Biophysics Unit, and Pathology, Princess Margaret Hospital, B. C. Cancer Research Centre, Vancouver, Toronto, Ontario M4X 1K9, Canada (59,139) BritishColumbiaVSZ lL3,Canada(509,647) HANSH ERWEIJERTN, O Institute of Applied DONALDP. EVENSONO, lson Biochemistry Radiobiology and Immunology, 2280 HV Laboratories, Department of Chemistry, Rijswijk, and Daniel den Hoed Cancer South Dakota State University, Brookings, Center, Rotterdam, The Netherlands (631) South Dakota 57007 (401) RYUJHI ~olrs~mvS~eoct,i on of Cancer Biology, JOHNJ . FAWCETTL,i fe Sciences Division, Los Radiation Oncology Center, Washington Alamos National Laboratory, University of University School of Medicine, St. Louis, Missouri 63108 (325, 353) California, Los Alamos, New Mexico 87545 (377) HOLGERH OEHND, epartment of Human Genetics, University of Wurzburg Biocenter, OSKARS . FRANKFURTG,'r ace Cancer Drug Am Hubland, 87 Wurzburg, Federal Republic Center, RosweU Park Memorial Institute, Buf- of Germany (185) falo, New York 14263 (13, 299) MARIANNHE. HOFLANDC,e ll Biology Group, MACKJ . FULWYLELRa, boratory for Cell Los Alamos National Laboratory, Los Analysis, Department of Laboratory Alamos, New Mexico 87545 (89) Medicine, University of California. San Fmn- cisco, California 94143 (613) PAULK ARLH OW, Zynaxis Cell Science, Inc., Malvern, Pennsylvania 19355 (469) DAVIDW . GALBRAITHD,e partment of Plant Sciences, University of Arizona, 'hcson, BRUCED . JENSENZ, ynaxis Cell Science, Inc., Arizona 85721 (527, 549) Malvern, Pennsylvania 19355 (469) ELIOG EIDOI, n,I stituto Nazionale per la Ricer- RICHARDJ ONKERT,N O Institute of Applied ca sul Cancro, Laboratoriod i Biofisica, 16132 Radiobiology and Immunology, 2280 HV Genova, Italy (149) Rijswijk, The Netherlands (631) JOHANNEGSE RDES,D ivision of Molecular CARLH . JUNE, Immune Cell Biology Program, Immunology, Forschungsinstitut Borstel, Naval Medical Research Institute, Bethesda, D-2061 Borstel, Federal Republic of Germany Maryland 20814 (37) (217) JANK APUSCINSCKanI,c er Research Institute of WALTEGR IARETTIIS, T, lstituto Nazionale per the New York Medical College at Valhalla, la Ricerca sul Cancro, Laboratorio di Valhalla, New York 10595 (655) Biofisica, 16132 Genova, Italy (149) MAREKK IMMEL,'I nvestigative Cytology JOEW . GRAYB, iomedical Sciences Division, Laboratory, Memorial Sloan-Kettering Lawrence Livermore National Laboratory, Cancer Center, New York, New York 10021 Livermore, California 94550 (207) (249) 'Present address Oncology Laboratory, Cedars Medical Center, Miami, Florida 33136. *Present uddess: Department of Statistics, Rice University, Houston, Texas 77251. CONTRIBUTORS xiii AWTARh s m , D epartment of Oncology, MARY J. O’CONNELAL,n alyticalC ytology Unit, University of Miami School of Medicine, Department of Pathology, University of Miami, Florida 33101 (121,491) Rochester Medical Center, Rochester, New York 14642 (501) MANFREDK UBBIESB, oehringer Mannheim Research Center, 8122 Penzberg, Federal P E ~LY. O LIVE,M edical Biophysics Unit, Republic of Germany (185) B. C. Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada (509) WEN-LINK uo, Biomedical Sciences Division, Lawrence Livermore National Laboratory, FRIEDRICOHT IQ Fachklinik Hornheide, D4WO Livermore, California 94550 (207) Miinster, Federal Republic of Germany (105) JUDITHL AFFIND. epartment of Microbiology DAN PINKELB, iomedical Sciences Division, and Immunology, Albany Medical College, Lawrence Livermore National Laboratory, Albany, New York 12208 (271) Livermore, California 94550 (383) JPROEN K. LARSENT, he Finsen Laboratory, ALANP OLLACKD,e partment of Radiation Rigshospitalet, University Hospital, DK-2100 Therapy, U.T.1M.D. Anderson Cancer Center, Copenhagen, Denmark (227) Houston, ’bas 77030 (19, 315) JOHN M. LEW, Department of Microbiology MARTINp bar, Departmento f Human Genetics, and Immunology, Albany Medical College, University of Wiirzburg Biocenter, Am Albany, New York 12208 (271) Hubland, 87 Wiirzburg, Federal Republic of Germany (185) ’Ikoaslls M. MCHUGHD, epartment of Labora- tory Medicine, University of California, San PETERS . RABINOVITCHD,e partment of Francisco, California 94143 (613) Pathology, School of Medicine, University of Washington, Seattle, Washington 98195 (37, MERYLJE. MELNICOPZPy, naxis Cell Science, 185) Inc., Malvern, Pennsylvania 19355 (469) FRANK A. RAY, Life Sciences Division. Los A. DUSTYM ILLEFRr,ed Hutchinson Cancer Re- Alamos National Laboratory, University of search Center, Seattle,W ashington 98104 (71) California. Los Alamos. New Mexico 87545 (369) ELIZABETAH. M USGROVGEa, rvan Institute of Medical Research, St. Vincent’s Hospital, JAYE . REEDERA, nalytical Cytology Unit, Darlinghurst, Sydney, N.S.W. 2010, Australia Department of Pathology, University of (59) Rochester Medical Center, Rochester, New York 14642 (501) KEES NOOTERT, NO Institute of Applied Radiobiology and Immunology, 2280 HV JOSEPH L. Ron Ron, Section of Cancer Biology, Rijswijk, The Netherlands (631) Radiation Oncology Center, Washington University School of Medicine, St. Louis, MICHAENLU =, GSF, Oesellschaft fiir Strahlen- Missouri 63108 (325, 353) und Umweltforschung, Institut fur Bio- physikalische Strahlenforschung, D-6000 HOWARDM . SHAPIROH, oward M. Shapiro, Frankfurt-am-Main, Federal Republic of Ger- M.D., P.C, West Newton. Massachusetts02165 many (149) (25) XiV CONTRIBUTORS K~STESNK ARSTADD, epartment of Biophysics, Laboratory, Livermore, California 94550 Institute for Cancer Research. Montebello, (363.631) 0310 Oslo, Norway (519) MARTINVA NDERLAAN.B iomedical Sciences SUE E. SLEZAKZ, ynaxis Cell Science, Inc.. Division, Lawrence Livermore National Malvern, Pennsylvania 19355 (469) Laboratory, Livermore, California 94550 HARALD B. STEEND, epartment of Biophysics, (207) Institute for Cancer Research, Montebello, LARSV INDF@VD, epartments of Hematology L 0310 Oslo, Norway (519) and Oncology ONK,R igshospitalet. Univer- sity Hospital, DK-2100 Copenhagen, Den- JOHNA . STEINJKAMP, Cell Biology Group, Los Alamos National Laboratory, Los Alamos, mark (127) New Mexico 87545 (199,305) JAN W. M. VISSERR, adiobiological Institute ANITAP. STEVENSOcNe.l l Biology Group, Los TNO, Rijswijk, The Netherlands (451) Alamos National Laboratory, Los Alamos, New Mexico 87545 (89) PETBR DE MUM. RadiobiologicalI nstitute, TNO, Rijswijk. The Netherlands (451) CABLETOCN. STEWARTL, aboratory of Flow Cytometry, ROswell Park Memorial Institute, Buffalo, New York 14263 (411,427) LEONL . WHBELESSJR, , Analytical Cytology Unit, Department of Pathology, University R~CHARAD. ' Ikiow, RATCOM. Inc, Miami, of Rochester Medical Center, Rochester. New Florida 33193 (111) York 14642 (501) JERRYT. '~ORNTHWAITE, Immuno-Oncology MARKE . WILDERC, ell Biology Group, Los Laboratories, Baptist Hospital of Miami, Alamos National Laboratory, Los Alamos, Miami, Florida 33176 (111) New Mexico 87545 (89) ROBERT A. "~EZY,B iochemistry and Biophysics WILLUMD. WRIGHTS, ection of Cancer Biology, Group, Los Alamos National Laboratory, Los Radiation Oncology Center, Washington Alamos. New Mexico 87545 (89, 97, 305) University School of Medicine, St. Louis, FRANK ~GANOSC. ancer Research Institute of Missouri 63108 (325, 353) the New York Medical College at Valhalla, Valhalla, New York 10595 (249) CLARICEM . Y~NTSCHJ., J. MacIsaac Flow Cyto- metrylsorting Facility, Bigelow Laboratory BAREMU'R USK, Biomedical Sciences Division, for Ocean Sciences, West Boothbay Harbor, Lawrence Livermore National Laboratory, Maine 04575 (613) Livermore, California 94550 (363,383) RICHARJD. Z m ,D epartment of Pathology, GERV AN DEN ENGHB. iomedical Sciences Divi- Henry Ford Hospital, Detroit, Michigan sion. Lawrence Livermore National 48202 (1) Progress in cell biology has been closely associated with the development of quantitative analytical methods applicable to individual cells or cell organelles. Three distinctive phases characterize this development. The first started with the introduction of microspectrophotometry, microfluorometry, and microinterferometry. These methods provided a means to quantitate various cell constituents such as DNA, RNA, or protein. Their application initiated the modern era in cell biology, based on quantitative- rather than qualitative, visual - cell analysis. The second phase began with the birth of autoradiography. Applications of autoradiography were widespread and this technology greatly contributed to better understanding of many functions of the cell. Especially rewarding were studies on cell reproduction; data obtained with the use of autoradiography were essential in establishing the concept of the cell cycle and generated a plethora of information about the proliferation of both normal and tumor cells. The introduction of flow cytometry initiated the third phase of progress in methods development. The history of flow cytometry is short, with most ad- vances occurring over the past 15 years. Flow cytometry (and, associated with it electronic cell sorting) offers several advantages over the two earlier methodologies. The first is the rapidity of the measurements. Several hundred, or even thousands of cells can be measured per second, with high accuracy and reproducibility. Thus, large numbers of cells from a given population can be analyzed and rare cells or subpopulations detected. A multitude of probes have been developed that make it possible to measure a variety of cell constituents. Because different constituents can be measured simultaneously and the data are recorded by the computer in list mode fashion, subsequent bi- or multivariate analysis can provide information about quantitative relationships among con- stituents either in particular cells or between cell subpopulations. Still another advantage of flow cytometry stems from the capability for selective physical sort- ing of individual cells, cell nuclei, or chromosomes, based on differences in the variables measured. Because some of the staining methods preserve cell viability and/or cell membrane integrity, the reproductive and immunogenic capacity of the sorted cells can be investigated. Sorting of individual chromosomes has already provided the basis for development of chromosomal DNA libraries, which are now indispensable in molecular biology and cytogenetics. Flow cytometry is a new methodology and is still under intense development, improvement, and continuing change. Most flow cytometers are quite complex and not yet user friendly. Some instruments fit particular applications better than others, and many proposed analytical applications have not been extensively tested on different cell types. Several methods are not yet routine and a certain degree xv xvi PREFACE of artistry and creativity is often required in adapting them to new biological material, to new applications, or even to different instrumentd esigns. The methods published earlier often undergo modifications or improvements. New probes are frequently introduced. This volume represents the first attempt to compile and present selected flow cytometric methods in the form of a manual designed to be of help to anyone interested in their practical applications. Methods having a wide immediate or potential application were selected, and the chapters are written by the authors who pioneered their development, or who modified earlier techniques and have extensive experience in their application. This ensures that the essential details are included and that readers may easily master these techniques in their laboratories by following the described procedures. The selection of chapters also reflects the peculiarity of the early phase of method development referred to previously. The most popular applications of flow cytometry are in the fields of immunology and DNA content-cell cycle analysis. While the immunological applications are now quite routine, many laboratories still face problems with the DNA measurements, as is evident from the poor quality of the raw data (DNAf requency histograms) presented in many publications. We hope that the descriptions of several DNA methods in this volume, some of them individually tailored to specific dyes, flow cytometers, and material (e.g., fixed or unfixed cells or isolated cell nuclei from solid tumors), may help readers to select those methods that would be optimal for their laboratory setting and material. Of great importance is the standardizationo f the data, which is stressed in all chapters and is a subject of a separate chapter. Some applicationso f flow cytometry included in this volume are not yet wide- ly recognized but are of potential importance and are expected to become widespread in the near future. Among these are methods that deal with fluores- cent labeling of plasma membrane for cell tracking, flow microsphere im- munoassay, the cell cycle of bacteria, the analysis and sorting of plant cells, and flow cytometric exploration of organisms living in oceans, rivers, and lakes. Individual chapters are designed to provide the maximum practical informa- tion needed to reproduce the methods described. The theoretical bases of the methods are briefly presented in the introduction of most chapters. A separate section of each chapter is devoted to applicability of the described method to different biological systems, and when possible, references are provided to ar- ticles that review the applications. Also discussed under separate subheads are the critical points of procedure, including the experience of the authors with dif- ferent instruments, and the appropriate controls and standards. Typical results, often illustrating different cell types, are presented and discussed in the “Results” section. The “Materials and Methods” section of each chapter is the most exten- sive, giving a detailed description of the method in a cookbook format. Flow cytometry and electronics orting have already made a significant impact