SPRINGER LAB MANUALS

Springer-Verlag Berlin Heidelberg GmbH

Rochelle A. Diamond Susan DeMaggio (Eds.)

In Living Color Protocols in Flow Cytometry and Cell Sorting

With 199 Figures and 26 Tables

Springer

ROCHELLE A. DIAMOND

Member of the Professional Staff California Institute of Technology Division of Biology 1200 E. California Blvd. Pasadena, CA 91125, USA Tel: 626 395-4947 Fax: 626 449-0756 E-mail: diamond@its.caltech.edu

ISBN 978-3-642-62978-5

Library of Congress Cataloging-in-Publication Data

SUSAN DEMAGGIO

MS BSMT (ASCP)CQ

Optical Biology Core Facility Developmental Biology Center University of California Irvine, CA 92687-2275, USA Tel: (949) 824-4110 Fax: (949) 824-3571 E-mail: suedemag@uci.edu

In living color: protocols in flow cytometry and cell sorting; with 26 tables I Rochelle A. Diamond; Susan DeMaggio (ed.).- Berlin; Heidelberg; New York; Barcelona; Hong Kong ; London ; Milan ; Paris ; Singapore ; Tokyo : Springer, 2000

(Springer lab manual) ISBN 978-3-642-62978-5 ISBN 978-3-642-57049-0 (eBook) DOI 10.1007/978-3-642-57049-0

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other way, and storage in data banks. Du­plication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permissions for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copy­right Law.

©Springer-Verlag Berlin Heidelberg 2000 Originally published by Springer-Verlag Berlin Heidelberg New York in 2000

The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such in­formation by consulting the relevant literature.

Production: PRO EDIT GmbH, 61126 Heidelberg, Germany Cover design: design & production GmbH, 69121 Heidelberg, Germany Typesetting: Mitterweger & Partner GmbH, 68723 Plankstadt, Germany SPIN: 10680098 27/3136/So 5 4 3 2 1 0

Acknowledgements

The Editors wish to thank the generous encouragement and sup­port of many people during the process of preparing this manual. Dr. Peter J. Bryant, Director of the Developmental Biology Cen­ter, and Dr. Michael Cahalan, Director of the Optical Biology Core Facility at the University of California, Irvine, who provided the allowance of time necessary to complete this project. This is especially true of Dr. Ellen Rothenberg at Caltech, who deserves a special thank you for her enthusiasm, advice, and personal kidness towards this, project. Without these peoples generosity this manual would not exist.

We would also like to thank all the authors who so generously offered their protocols, and spent many hours preparing their individual manuscripts for inclusion in the manual, and a special thanks to our preface author, Dr. Harry Crissman. Without the assistance of Paul Robinson and Steve Kelley and the Purdue Cytometry Listserv, we would not have been able to recruit authors and research new protocols for inclusion in the text. The Table of Contents illustrates the number and caliber of each of these authors. We would like to also thank those who prepared appendix materials and vendors who suppor­ted the printing of color plates for the illustration of the protocols.

Technical assistance for typing, computer problems, and se­cretarial tasks was invaluable. Much appreciation is expressed to Bob Turing of the Caltech Graphic Arts Facility and Stephanie Canada for their help in downloading unusual files and scanning figures. Our thanks especially go to Rochelle's personal assistant, Catherine Springer, and to Amanda and Ronald Diamond for loan of a laptop computer to make life easier. We want to thank Barbara Belmont for her help proofreading and in preparation of the glossary as well as personal assistance with software support

VI Acknowledgements

and computer problems, her cheerful encouragement, and din­ner a few nights, too!!

I, Sue, would like to thank my family left at home, Mark and Laura DeMaggio for their understanding of the long hours at work and the late and missing meals. And Rochelle, for being a wonderful compliment to my skills. She is a delight to work with and we seemed to be a great team. She also rewarded me with wonderful meals on long evenings in Pasadena working on the manuscript.

I, Rochelle, could never have taken on such an endeavor with­out Sue's organizational and FTP skills. I never realized how much work goes into one of these projects, even when you think it is done. For my part, I dedicate this work to my family and friends whose encouragement keeps me flowing.

Pasadena/Irvine September 1999

THE EDITORS

Guest Preface

Advances in the field of cell biology have always been closely related to the development of quantitative analytical methods that can be applied to individual cells or cell organelles. Almost from the early stages following the invention of the microscope, the investigator has been keenly interested in obtaining informa­tion on the functionality of single cells and how cells perform under different sets of experimental conditions. Although cells could be viewed in the microscope for a few hundred years, only since the relatively recent application of autoradiography did we come to realize that, although cells may visually appear very much alike, they are quite different in their functional capacity. The quest to understand these differences in a cell population lead to a new series of techniques for labeling and quantitating DNA content and similar approaches have driven the develop­ment of methods for analyzing various other cellular properties.

The development of new analytical techniques follows the age old pattern of applying successes of the past with current inno­vation, logic and new biological information. Results from auto­radiography expanded the concept of the cell cycle from inter­phase and mitosis to the more definitive GO/GI, Sand G2/M phases. This new knowledge lead to the development of techno­logy to measure and analyze various parameters related to the cell cycle. The results of such studies indicated that the dynamic nature of cycle progression, cell maturation and other physio­logical processes, involves changes in the size, the shape and function of the various cell organelles that are rapid and under well coordinated, temporal regulation. From numerous observa­tions it has become clear that a better understanding of cell phy­siology can only be obtained by the measurement of as many functional properties as possible in single cells. The concept is now well-founded and has been more accurately described

VIII Guest Preface

as the multiparameter analysis approach. From such an analysis it is possible to obtain a more accurate understanding about the normal operations of the cell machinery and thereby be better positioned to assess changes that occur under experimental or pathological conditions.

When attempting to develop new analytical techniques, one quickly realizes the limitations of our physical attributes to make rapid quantitative measurements. Under a microscope one can­not distinguish a G 1 from a G2 phase cell stained with a fluo­rescent dye, although there is a relatively two fold difference in intensity. For obvious reasons then, investigators have pursued the parallel development of cell probes and instrumen­tation that can measure, quantitatively, particular markers of interest. Although significant strides have been achieved, it is clear that much of the methodology is still quite complex. Most instrumentation is still complicated and not always user friendly nor universal in their application to all types of measure­ments. Some methods are routine and well-tested, while others require well defined conditions and some careful consideration in adapting them to different biological material. It is important to consider all the pertinent aspects when undertaking the applications of new analytical procedures and locating the relevant information in a detailed, but easy to grasp context, can be of considerable benefit.

The editors developed this manual with consideration of the needs of the beginner and the more experienced cytometrist at­tempting to expand both their range of applications and/or their area of investigations. The applications, both immediate and po­tential, have been selected and presented by authors who have in-depth knowledge of the well controlled use and limitations of the various techniques. This compilation of chapters is intended to save the investigator from the tedious task of searching the literature for those details on the particular analytical technique that are, in fact, often lacking in the journal publications.

The manual is divided into nine sections with a detailed ap­pendix. The early chapters provide the fundamentals on the op­eration of a flow cytometer, data display and analysis. These de­tails will guide the reader into limitations and realistic expecta­tions of the instrument and interpretation of results from com­plicated measurements. Sample preparation for single and multiple parameter analysis, an important, critical consideration

Guest Preface IX

in any experiment, is addressed in considerable detail, along with labeling and multi-color fluorescence analysis of cell sur­face markers. Analysis of reporter genes, a relatively new appli­cation, particularly adapted to cytometry while cell tracking methods have become more popular for determining the ac­countability of particular subpopulations of interest. Many stud­ies continue to include cell cycle phase-specific as well as RNA content determinations, and a section is provided on appropri­ate nucleic acid labeling and analysis for both plant and animal cells and the use of selected fluorochromes for identifying cells in apoptosis. Techniques for analysis of cell physiology involving probes for intracellular pH, Ca determinations and cytoenzyme systems provide the reader with approaches for examining the functionality of single cells. Recovery of viable cells by physical sorting remains of interest to many investigators, but actually employing the technique is not an easy task. However, the man­ual provides a straight forward approach for performing sorting of viable cells under sterile conditions. The successful and safe operation of the core facility is presented in some detail by in­dividuals who have really researched the subject and obtained the input of a number of facility operators. The appendix con­tains a wealth of valuable information for quick reference to the availability and the commercial sources that can provide lasers, fluorescent compounds, computer software and specific anti­bodies. In general, the editors have made every attempt to compile a very comprehensive manual that should be extremely useful to the reader. They have selected the various sections based on their long standing experience in the field of cytometry and on the questions and analytical problems that have been brought to them by investigators wishing to analyze a wide range of biological material and often times using probes that require much adaptation of the methodology.

HARRY CRISSMAN

Contents

Section 1

A Palette of Living Colors ROCHELLE DIAMOND ................................ 3

The Colorful History of Flow Cytometry SUSAN DEMAGGIO. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Section 2

Getting Started ROCHELLE DIAMOND

Chapter 1 How the FACSCalibur and FACS Vantage Work and Why It Matters

13

TOM FREy.......................................... 15

Chapter 2 How Flow Cytometers Work - and Don't Work HOWARD SHAPIRO .................................. 39

Chapter 3 Flow Cytometry Standard (FCS) Data File Format LARRY SEAMER ..................................... 57

Chapter 4 Options for Data Display SUSAN DEMAGGIO AND ROCHELLE DIAMOND .......... 62

XII Contents

Chapter 5 Guidelines to Improve Flow Cytometry Data Display and Interpretation WENDY SCHOBER-DITMORE AND DOROTHY LEWIS 71

Chapter 6 Data Management and Storage DAVID M. CODER. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 76

Chapter 7 Coloring up: A Guide to Spectral Compensation ROCHELLE A. DIAMOND. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 92

Chapter 8 Quality Control Guidelines for Research Flow Cytometry ROCHELLE A. DIAMOND. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 98

Section 3

Sample Preparation and Cell Surface Staining ROCHELLE A. DIAMOND .............................. 109

Chapter 1 Cell Preparation and Enrichment for FCM Analysis and Cell Sorting ROCHELLE A. DIAMOND, HUA WANG, FEI CHEN,

AND MINDY WILKE-DOUGLAS ......................... 111

Chapter 2 The Basics of Staining for Cell Surface Proteins ALICE L. GIVANS .................................... 142

Chapter 3 Phenotypic Analysis Using Five-Colour Immunofluorescence and Flow Cytometry ANDY BEAVIS ....................................... 165

Chapter 4 Detection of Cell Surface Antigens Using Antibody­Conjugated Fluorospheres and Flow Cytometry ANDY BEAVIS ....................................... 175

Contents XIII

Chapter 5 Gel Microdrop Encapsulation for the Frugal Investigator DAVID C. MCFARLAND AND GARY DURACK ............ 184

Section 4

Reporter Genes and Cell Trackers ROCHELLE A. DIAMOND .............................. 197

Chapter 1 Flow Cytometric Analysis of GFP Expression in Mammalian Cells STEVEN R. KAIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 199

Chapter 2 FACS-Gal: Flow Cytometric Assay of ~-galactosidase in Viable Cells STEVEN FIERING

Chapter 3

227

Utilization of a Bicistronic Expression Vector for Analysis of Cell Cycle Kinetics of Cytotoxic and Growth-arrest Genes NICHOLAS E. POULOS, ERIC STANBRIDGE,

AND SUSAN DEMAGGIO .............................. 259

Chapter 4 NGFR as a Selectable Cell Surface Reporter G. RAGHU, M. MUNN, S. W. PARK, C-H PAN, AND A. DAYN 267

Chapter 5 Assessment of Cell Migration in vivo Using the Fluorescent Tracking Dye PKH26 and Flow Cytometry ANDY BEAVIS

Chapter 6 In vivo Biotinylation for Cell Tracking and Survival/Lifespan Measurements

276

KENNETH A. AULT AND CATHY KNOWLES ............. 284

Chapter 7 Enrichment and Selection for Reporter Gene Expression with MACSelect MINDY WILKE-DOUGLAS AND UTE BEHRENS-JUNG ...... 291

XIV Contents

Chapter 8 Use of PKH Membrane Intercalating Dyes to Monitor Cell Trafficking and Function REBECCA PO ON, BETSY M. OHLSSON-WILHELM,

BRUCE BAGWELL, AND KATHARINE A. MUIRHEAD ...... 302

Section 5

Flow Cytometry of Nucleic Acids ROCHELLE A. DIAMOND. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 355

Chapter 1 RNA Content Determination Using Pyronin Y HOWARD M. SHAPIRO ............................... 357

Chapter 2 DNA Content Determination DEREK DAVIES AND CLARE HUGHES .................. 361

Chapter 3 Dead Cell Discrimination DEREK DAVIES AND CLARE HUGHES .................. 372

Chapter 4 Detecting Apoptosis DEREK DAVIES AND INGRID SCHMID 377

Chapter 5 Chromosome Preparation - Polyamine Method DEREK DAVIES AND CLARE HUGHES .................. 385

Chapter 6 'D-Flowering' - The Flow Cytometry of Plant DNA JOHN MOTLEY, K. YOKOYA, AND A.V. ROBERTS ......... 391

Chapter 7 Flow Cytometry Analysis of Marine Picoplankton DOMINIQUE MARIE, N. SIMON, L. GUILLON,

F. PARTENSKY, AND D. VAULOT ....................... 421

Chapter 8 Yeast DNA Flow Cytometry WENDY SCHOBER-DITMORE AND JEFF BACHANT ....... 455

Contents XV

Section 6

Intracellular Physiological and Antibody Probes ROCHELLE A. DIAMOND. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 461

Chapter 1 Calcium Flux Protocol: INDO-1 Stained Thymocytes GEOFFREY W. OSBORNE AND PAUL WARING 463

Chapter 2 Intracellular pH Measured by ADB MICHAEL FOX ....................................... 472

Chapter 3 Measuring Intracellular pH Using SNARF-l MICHAEL FOX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 481

Chapter 4 FACS Analysis of Ph and Apoptosis GRANT W. MEISENHOLDER, JUDY NORBERG,

AND ROBERTA A. GOTTLIEB .......................... 490

Chapter 5 Measurement of Ligand Acidification Kinetics for Adherent and Non-Adherent Cells SHEREE LYNN RYBACK AND ROBERT F. MURPHY ....... 496

Chapter 6 Intracellular Antigen Detection by Flow Cytometry INGRID SCHMID . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 524

Chapter 7 Flow Cytometric Measurement of Nuclear Matrix Proteins JOSEPH R. DYNLACHT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 532

Chapter 8 CellProbe Flow Cytoenzymology SUSAN DEMAGGIO ................................... 539

Chapter 9 Intracellular Cytokine Detection MARY A. YUI ........................................ 552

XVI Contents

Section 7

Electronic Cell Sorting ROCHELLE A. DIAMOND. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 563

Chapter 1 Creating Standard and Reproducible Sorting Conditions MARTY BIGOS, RICHARD T. STOVEL, AND DAVID R. PARKS 565

Chapter 2 Sterilization for Sorting STEVEN MERLIN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 572

Chapter 3 High Speed Cell Sorting Using the Cytomation CICERO® and MoFlo® Systems PETER LOPEZ ....................................... 577

Chapter 4 Purification of Mouse Fetal Liver Hematopoietic Stem Cells SEAN J. MORRISON .................................. 585

Chapter 5 Methods for Preparing Sorted Cells as Monolayer Specimens ROBERT C. LEIF ..................................... 592

Chapter 6 Fluorescent in Situ Hybridization Analysis on Cells Sorted onto Slides KAREN CHEW AND ROBERT C. LEIF

Section 8

Core Facilities

620

ROCHELLE A. DIAMOND .............................. 633

Chapter 1 Core Facility Management STEVEN MERLIN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 635

Chapter 2 Funding for Core Facilities LARR Y A. SKLAR .................................... 642

Chapter 3 Biosafety in the Flow Cytometry Laboratory INGRID SCHMID ..................................... 655

Contents XVII

Section 9

Unlocking the Spectrum of Possibilities ROCHELLE A. DIAMOND. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 669

Appendices

GFP STEVEN KAIN

Fluorescent Protein Newsgroup

677

STEVEN KAIN ....................................... 679

Anderson's Timesaving Comparative Guides 681

ARC Lamp Flow Cytometers JEFF HARVEY ....................................... 683

CompCyte Laser Scanning Cytometer SUSAN DEMAGGIO ................................... 691

Coulter Flow Cytometry Systems SUSAN DEMAGGIO ................................... 697

Cytomation Products for Upgrading Flow Cytometers SUSAN DEMAGGIO ................................... 701

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 703

Lasers in Flow Cytometry PAUL GINOUVES .................................... 715

Resources for Reagents ............................... 731

Flow Cytometry Internet Access and W orId Wide Web Sites of Interest . . . . . . . . . . . . . . . .. 732

User Groups Around the Country ..................... 736

Commercial Suppliers for Flow Related Products 739

Data Analysis for Cell Census Plus ™ System REBECCA POON ..................................... 754

Monoclonal Antibody Production GARY WOO ......................................... 776

Subject Index ....................................... 789

list of Contributors

AULT, KENNETH A.

Maine Medical Center Research Institute, MMCRI, 125 John Roberts Rd., Suite 8, South Portland, ME 04106, USA

BACHANT, JEFF

Dept. of Biochemistry, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA

BAGWELL, BRUCE

Verity Software House, Inc., PO Box 247, 45A Augusta Rd., Topsham, ME 04086, USA

BEAVIS, ANDY

Flow Cytometry Core Facility, Princeton University, Department of Molecular Biology, Lewis Thomas Laboratory, Washington Road, Princeton, NJ 08544-1014, USA

BEHRENS-JUNG, UTE

Miltenyi Biotec GmbH, Friedrich-Ebert-Strasse 68, 51429 Bergisch Gladbach, Germany

BIGOS, MARTIN

Senior Scientific Programmer, Department of Genetics, Stanford University School of Medicine, Beckman B-007, Stanford, CA 94305, USA

CHEN, FEI

California Institute of Technology, Division of Biology, 1200 E. California Blvd., Pasadena, CA 91125, USA

CHEW, KAREN

University of California at San Francisco, Cancer Center, Room S435, 2340 Sutter Street, San Francisco, CA 94115, USA

xx List of Contributors

CODER, DAVID M.

University of Washington, School of Medicine, Dept. ofImmunology, Box 357650, Seattle, WA 98195-7650, USA

DAYN,ANDREW

Director of Development, Ingenex, Inc., 400 Oyster Point Blvd., Suite 505, South San Francisco, CA 94080-1921, USA

DAVIES, DEREK

FACS Laboratory (Room 1Bl), Imperial Cancer Reserach Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

DEMAGGIO, SUSAN

Optical Biology Core Facility, Developmental Biology Center, University of California, Irvine, CA 92687-2275, USA

DIAMOND, ROCHELLE A.

California Institute of Technology, Division of Biology, 1200 E. California B., Pasadena, CA 91125, USA

DURACK,GARY

Biotechnology Center Flow Cytometry Facility, University of Illinois at Urbana-Champaign, 231 ERML, 1201 W. Gregory Dr., Urbana, IL 61801, USA

DYNLACHT, JOSEPH R.

Indiana University School of Medicine, Dept. of Radiation Oncology, Indiana Cancer Pavilion, RT 041, 535 Barnhill Dr., Indianapolis, IN 46202, USA

FIERING, STEVEN

Microbiology Department, Dartmouth Medical School, 6 West Boswell, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA

FOX, MICHAEL H.

Professor, Radiological Health Sciences, Chairman, Graduate Program in Cell and Molecular Biology, Colorado State Univ., Fort Collins, CO 80523 -1673, USA

FREY, TOM

Becton Dickenson, Becton Dickinson Immunocytometry Systems, 2350 Qume Road, San Jose, CA 95l31, USA

GINOUVES, PAUL

Coherent, Laser Group, 5100 Patrick Henry Dr., Santa Clara, CA 95054, USA

GIV ANS, ALICE L.

List of Contributors XXI

Englert Cell Analysis Laboratory of the Norris Cotton Cancer Center, Dartmouth Medical School, Dept. of Physiology, Borwell Building, Lebanon, NH 03756-0001, USA

GOTTLIEB, ROBERTA

The Scripps Research Institute, 10550 N. Torrey Pines Rd. NX7, La Jolla, CA 92037, USA

GUILLOU, LAURE

CNRS-UPR 9042, Station Biologique de Roscoff, Place Georges Teissier, 29680 Roscoff, France

HARVEY, JEFF

Bio-Rad Laboratories Inc., Flow Cytometry, 4000 Alfred Nobel Dr., Hercules, CA 94547, USA

HUGHES, CLARE

FACS Laboratory (Room IBI), Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

KAIN, PH.D., STEVEN R.

CIon tech Laboratories Inc, Cell Biology Group, 1020 E. Meadow Circle, Palo Alto, CA 94303-4230, USA

KNOWLES, CATHY

Maine Medical Center Research Institute, MMCRI 125 John Roberts Rd., Suite 8, South Portland, ME 04106, USA

KRUEGER, TOM

Coulter, Mail Code: 42 - B01, P.O. Box 169015, Miami, FL 33116-9015, USA

LEWIS, DOROTHY

Baylor College of Medicine, Department of Microbiolgoy and Immunology M91O, One Baylor Plaza, Houston, TX 77030, USA

LEIF, ROBERT C.

Vice President, Newport Instruments, 5648 Toyon Rd, San Diego, CA 92115-1022, USA

XXII List of Contributors

LOPEZ, PETER A.

Cytomation, Inc., 4850 Innovation Dr., Fort Collins, CO 80525, USA

MARIE, DOMINIQUE

CNRS-UPR 9042, Station Biologique de Roscoff, Place Georges Teissier, 29680 Roscoff, France

MCFARLAND, DAVID C.

Howard Hughes Medical Institute Flow Cytometry Facility, 807 Light Hall, Vanderbilt University Medical Center, Nashville, TN 37232-0295, USA

MEISENHOLDER, GRANT W.

Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA

MERLIN, STEVEN

Flow Cytometry Facility, Hospital for Special Surgery, Research Bldg, Room 312, 535 E. 70th St., New York, NY 10021, USA

MORRISON, SEAN J.

Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, 3215 Comprehensive Cancer Center, 1500 E. Medical Center Drive, Ann Arbor, MI, 48109-0934 USA

MOTTLEY, JOHN

Cellular and Molecular Biology Research Unit, Department of Life Sciences, University of East London, Romford Road, London E15 4LZ, UK

MUNN, MALCOLM

Chromaprobe Inc., 897 Independance Ave 4C, Mountain View, CA 94043, USA

MURPHY, ROBERT F.

Department of Biological Sciences, Carnegie Mellon University, 440 Fifth Avenue, Pittsburgh, PA 15213, USA

NORBERG, JUDITH

Research Flow Cytometry Lab, V A San Diego Medical Care Health System, 3350 La Jolla Village Dr. VI5I, La Jolla, CA 92161, USA

List of Contributors XXIII

OSBORNE, GEOFFREYW.

Flow Cytometry Laboratory, EM/HISTO/FACS Unit, John Curtin School of Medicine, Australian National University, PO Box 334, National Australian Capital, Canberra, 2601, Australia

PAN, ALISON CHINHUEI

PPD Discovery, Inc. 1505 O'Brien Drive, Suite 13, Menlo Park, CA 94025-1435, USA

PARK, SUK

University of California, Davis School of Medicine, Davis, CA 95616, USA

PARKS, DAVID R.

Department of Genetics, Stanford University School of Medicine, Beckman B-007, Stanford, CA 94305 - 5125, USA

PARTENSKY, FREDERIC

CNRS-UPR 9042, Station Biologique de Roscoff, Place Georges Teissier, 29680 Roscoff, France

POON, REBECCA

Sigma Chemicals, R&D Department 47,3600 S. 2nd St., St. Louis, MO 63118, USA

POULOS, NICHOLAS

Southside Neurosurgical Associates, 183 S. Main St., Danville, VA 24541, USA

RAGHU, GANAPATHIRAMA

Discovery Research, Protein Science Dept., INCYTE Pharmaceu­ticals, Inc., 3174 Porter Drive, Palo Alto, CA 94304, USA

ROBERTS, ANDREW V.

Cellular and Molecular Biology Research Unit, Department of Life Sciences, University of East London, Romford Road, London E15 4LZ, UK

RYBACK, SHEREE L.

Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA

XXIV List of Contributors

SCHMID, INGRID

Flow Cytometry Specialist, UCLA School of Medicine, Division of Hematology-Oncology, 10833 Le Conte Ave., Los Angeles, CA 90095, USA

SCHOBER-DITMORE, WENDY

MLT (ASCP), QCYM, FLow Cytometry Specialist, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030

SEAMER, LARRY

University of New Mexico, Albuquerque, NM 87131, USA

SHAPIRO, HOWARD

283 Highland Ave., West Newton, MA 02165-6044, USA

SIMON, NATALIE

CNRS-UPR 9042, Alfred Wegener-Institut fUr Polar- und Meeresforschung, Am Handelshafen 12, 27570 Bremerhaven, Germany

SKLAR, LARRY

Professor of Pathology, UNM Health Sciences Center, Cancer Research Facility Bldg. 229, UNM Health Science Center, Albuquerque, NM 87131, USA

STANBRIDGE, ERIC

Department of Microbiology and Molecular Genetics, Mail Code 4025 B235, B210 MedSci I, University of California, Irvine, CA 92687-2275, USA

STOVEL, RICHARD T.

Department of Genetics, Stanford University School of Medicine, Beckman B-007, Stanford, CA 94305-5125, USA

VAULOT, DANIEL

CNRS-UPR 9042, Station Biologique de Roscoff, Place Georges Teissier, 29680 Roscoff, France

WANG, HUA

Stowers Medical Research Institute, Caltech Division of Biology, 1200 E. California Blvd., Pasadena, CA 91125, USA

List of Contributors XXV

WARING, PAUL

Apoptosis Laboratory, Division of Immunology and Cell Biology, John Curtin School of Medicine, Australian National University, PO Box 334, National Australian Capitol Territory, 2601 Australia

WILKE-DOUGLAS, MINDY

Miltenyi Biotec, 251 Auburn Ravine Road, Suite 208, Auburn, CA 95603, USA

WOO, GARY

Amgen Inc., 1840 DeHavilland Dr., Thousand Oaks, CA 91320, USA

YOKOYA,KAZUTOMO

Cellular and Molecular Biology Research Unit, Department of Life Sciences, University of East London, Romford Road, London E15 4LZ, UK

YUI, MARY

California Institue of Technology, Division of Biology 156 - 29, 1200 E. California Blvd., Pasadena, CA 91125, USA