Table Of ContentMETHODS 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
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LESLIE WILSON
Department of Biological Sciences
University of California
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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
