Methods in Cell Biology

VOLUME 6 3Cytometry

Third Edition, Part A

Zbigniew Darzynkiewicz

Harry A. Crissman

J . Paul Robinson

Contents of Volume 64

xii

Contributors

xvii

Preface to the Third Edition

xxi

Preface to the Second Edition

xxv

Preface to the First Edition

xxi x

PART I Principles of Cytometry and General Method s

1 . A Brief History of Flow Cytometry and Sortin g

Myron R . Melamed

I. Introduction

3II. Instrumentation

4III. Applications

9References

1 3

2 . Principles of Flow Cytometry : An Overview

Alice L . Givan

I. Introduction

1 9II. The Illumination of a Particle

20III. Fluidics : Centering Particles in the Illuminating Beam

26IV. Collection of Light Signals from Particles

33V. From Light Signals to a Data File

37VI. From Data to Information

4 1VII. Sorting

44VIII. Conclusions

4 8References

4 8

3 . Laser Scanning Cytometry

Louis A . Kamentsk y

I. Introduction

5 1II. Background

5 2III. Description of the Instrument

5 5IV. The Utility and Operational Characteristics of Some Laser Scanning

62Cytometry List Mode Features

V. Utility of Solid Phase Cytometry for Cell Preparation

80

VI. Future Directions

8 2

References

8 4

4 . Principles of Confocal Microscop y

J. Paul Robinso n

I. Brief History of Microscope Development

8 9

II. Development of Confocal Microscopy

9 0

III. Image Formation in Confocal Microscopy

9 2

IV. Useful Fluorescent Probes for Confocal Microscopy

9 6

V. Applications of Confocal Microscopy

10 0

VI. Conclusions

105

References

105

5 . Optical Measurements in Cytometry : Light Scattering, Extinction ,

Absorption, and Fluorescenc e

Howard M. Shapiro

I. Introduction

10 8

II. Signal Processing Tasks in Flow Cytometry : An Overview

10 8

III. The Optical Signal: Interaction of Light with Cells

11 0

IV. Detection : Converting Optical Signals to Current

11 8

V. Electronics : Converting Current to Voltage

123

VI. Fluorescence Compensation and Logarithmic Amplification

124

VII. Peak Detection, Integration, and Pulse Width Measurement ; Triggering

127

VIII. Measurement Sensitivity : Changing Concepts and the Bottom Line

127

References

12 9

6 . Flow Cytometric Fluorescence Lifetime Measurement s

Harry A . Crissman and John A . Steinkamp

I. Introduction

13 1

II. Applications of the Technology

13 4

III. Cell Preparation and Staining

13 7

IV. Fluorescence Lifetime Flow Cytometry Instrumentation

13 8

V. Results

14 1

VI. Critical Aspects of the Technology

14 4

VII. Future Directions

14 6

References

14 7

7 . Principles of Data Acquisition and Display

Howard M. Shapiro

I. Introduction

14 9

II. Pulse Characterization Using Analog and Hybrid Circuits

15 0

III. Analog Data Display

154

IV. Analog-to-Digital Conversion

15 5

V. Pulse Characterization by Digital Signal Processing

15 9

VI. Data Storage and Display with Digital Computers

16 1

References

16 7

8 . Time as a Flow Cytometric Paramete r

Larry Seamer and Larry A . Skla r

I. Introduction

16 9

II. Historical Overview

17 1

III. Sample Mixing and Delivery

17 1

IV. Data Analysis

17 5

V. Applications

17 9

VI. Conclusions

18 1

References

18 1

9 . Protein Labeling with Fluorescent Probe s

Kevin L. Holmes and Larry M. Lantz

I. Introduction

185

II. Labeling of Proteins with Organic Fluorescent Dyes

186

III. Labeling of Proteins with Phycobiliproteins

194

IV. Conclusion

202References

202

PART II Cell Preparation

10 . Preparation of Cells from Bloo d

J . Philip McCoy, Jr.

I. Introduction

207

II. Collection, Transport, and Storage of Blood

20 8

III. Fixation and Preservation

21 0

IV. Separation of Erythrocytes from Leukocytes

21 0

V. Assessment of Cell Viability

21 1

VI. Staining

21 2VII. Summary

21 2Appendix 1

21 3Appendix 2

21 4Appendix 3

21 4References

21 4

11 . Cell Preparation for the Identification of Leukocytes

Carleton C . Stewart and Sigrid'. Stewar t

I. Introduction

21 8II. Antibodies

21 9III. Tandem Fluorochromes

234

IV. Cell Preparation and Staining Procedures

23 7V. Titering Antibodies

24 4VI. Solutions and Reagents

24 9References

25 0

12 . Strategies for Cell Permeabilization and Fixation in Detecting Surfac eand Intracellular Antigens

Steven K. Koester and Wade E . Bolto n

I. Introduction

25 3II. Application

25 4III. Materials and Methods

26 2IV. Concluding Remarks

26 6References

26 6

PART III Standardization, Quality Assuranc e

13 . Stoichiometry of Immunocytochemical Staining Reaction s

James W. Jacobberge r

I. Introduction

27 1II. Structure of Immunoglobulin G

27 3III. Cell Structure

274IV. Permeabilized Cell Structure

274V. Antibody-Antigen Reactions

28 1VI. Multiparametric Analyses

292VII. Summary

294References

29 5

14 . Standardization and Quantitation in Flow Cytometr y

Robert A . Hoffma n

I. Introduction

300II. General Issues

300III. Performance Characteristics-Dynamic Range, Linearity, Resolution,

302and Sensitivity

IV. Standardization and Calibration of Common Cytometry Measurements

31 2V. Examples of Applications Using Calibrated Measurements

33 0VI. Issues in Quantitation of Fluorochromes and Other Molecules

33 2References

33 4

PART IV Cell Proliferation

15 . Methods to Identify Mitotic Cells by Flow Cytometr y

Gloria Juan, Frank Traganos, and Zbigniew Darzynkiewicz

I. Introduction

34 3II. Materials

345

III. Cell Preparation and Staining

34 5

IV. Instruments

34 6

V. Critical Aspects of the Procedure

34 6

VI. Results and Discussion

34 7

VII. Comparison of Anti-H3-P Monoclonal Antibody with Other Markers of

35 0

Mitotic Cells

References

35 3

16 . Cell Cycle Kinetics Estimated by Analysis of Bromodeoxyuridin eIncorporatio n

Nicholas H . A . Terry and R . Allen White

I. Introduction

35 5

II. Applications

35 7

III. Materials

35 8

IV. Methods

36 0

V. Critical Aspects of the Procedure

36 9

References

37 2

17 . Flow Cytometric Analysis of Cell Division History Using Dilution ofCarboxyfluorescein Diacetate Succinimidyl Ester, a Stably Integrate d

Fluorescent Prob e

A. Bruce Lyons, Jhagvaral Hasbold, and Philip D . Hodgki n

I. Introduction and Background

376

II. Reagents and Solutions

37 8

III. Preparation and Labeling of Cells

37 8

IV. Gathering of Information Concurrent with Division

38 1

V. Analysis of Data

38 6

VI. Application of Carboxyfluorescein Diacetate Succinimidyl Ester to in Vitro

39 0

Culture of Lymphocyte sVII. Monitoring Lymphocyte Responses in Vivo

39 2

VIII. Antigen Receptor Transgenic Models

39 5

References

39 7

18 . Antibodies against the Ki-67 Protein : Assessment of the Growth Fraction

and Tools for Cell Cycle Analysis

Elmar Endl, Christiane Hollmann, and Johannes Gerdes

I. Introduction

399

II. Application

40 1

III. Materials and Methods

402

IV. Critical Aspects

40 8

V. Controls and Standards

40 9

VI. Examples of Results

41 0

References

414

19 . Detection of Proliferating Cell Nuclear Antige n

Jorgen K. Larsen, Göran Landberg, and Göran Roo s

I. Introduction

41 9II. Molecular Biology of Proliferating Cell Nuclear Antigen

42 0III. Methods for Immunochemical Detection and Quantification of

42 1Proliferating Cell Nuclear Antigen

IV. Results of Cytometric Analysis of Proliferating Cell Nuclear Antigen

42 3Expression

V. Applications in Toxicology, Pathology, and Oncology

42 6References

42 8

20 . Lymphocyte Activation Associated Antigen s

Andrea Fattorossi, Alessandra Battaglia, and Cristiano Ferlini

I. Introduction

43 3II. Methodological Aspects

43 7III. To Flow or Not to Flow for Assessing Lymphocyte Activation/

44 7Proliferation? And, If Yes, How Reliable Is Immunophenotyping ?

IV. Additional Approaches

45 3V. Concluding Remarks

45 5References

45 7

PART V Cell Death/Apoptosis

21. Analysis of Mitochondria during Cell Death

Andrea Cossarizza and Stefano Salviol i

I. Introduction

467II. Scientific Background

46 8III. Apoptosis and Mitochondria

470IV. Method

472V. Results

473VI. Pitfalls and Misinterpretation of the Data

476VII. Comparison with Other Methods

477VOL Reviews of the Applications

478IX. Biological and Biomedical Information

47 9X. Future Directions

48 0References

48 1

22 . Cytometry of Caspase s

Steven K. Koester and Wade E . Bolto n

I. Introduction

48 7II. Materials and Methods : Caspase Peptide Inhibitors and Methods to

49 0Monitor Response s

III. Results and Discussion

49 7References

502

23 . Analysis of Apoptosis in Plant Cells

Iona E . Wei r

I. Introduction

50 5

II. Apoptosis in Plants

50 6

III. Problems Associated with Analyzing Plant Cells Using Flow Cytometry

50 9

IV. Morphological Changes of Plant Cells

51 2

V. Physiological Changes during Apoptosis

520

VI. Conclusion

524

References

524

24 . Difficulties and Pitfalls in Analysis of Apoptosi s

Zbigniew Darzynkiewicz, Elzbieta Bedner, and Frank Tragano s

I. Introduction

527

II. Apoptotic Index May Not Be Correlated with Incidence of Cell Death

52 9

III. Difficulties in Estimating Frequency of Apoptosis by Analysis of DNA

53 1

Fragmentatio n

IV. The Lack of Evidence Is Not Evidence for the Lack of Apoptosis

53 2

V. Misclassification of Apoptotic Bodies or Nuclear Fragments as Single

53 3

Apoptotic Cells

VI. Apoptosis versus Necrosis versus " Necrotic Stage" of Apoptosis

53 5

VII. Selective Loss of Apoptotic Cells during Sample Preparation

53 7

VIII. Live Cells Engulfing Apoptotic Bodies Masquerade as Apoptotic Cells

53 8

IX. The Problems with Commercial Kits and Reagents

53 8

X. Cell Morphology Is Still the Gold Standard for Identification of Apoptotic

53 9

Cell sXI. Laser Scanning Cytometry : Have Your Cake and Eat It Too

54 1

References

54 4

PART VI Cell-Cell, Cell-Environment Interaction s

25 . Analysis of Cell Migration

Nicole Dodge Zantek and Michael S . Kinch

I. Introduction and Application

549

II. General Strategies to Measure Cell Migration

55 0

References

55 8

26 . Three-Dimensional Extracellular Matrix Substrates for Cell Cultur e

Sherry L. Voytik-Harbin

I. Introduction

56 1

II. Application

56 3

III. Methods

56 5

IV. Application of Intestinal Submucosa as a Three-Dimensional Extracellular

57 2

Matrix Substrat eV. Summary

57 6

References

578

27. Three-Dimensional Imaging of Extracellular Matrix and Extracellula r

Matrix-Cell Interactions

Sherry L. Voytik-Harbin, Bartlomiej Rajwa, and J . Paul Robinso n

I. Introduction

58 3II. Three-Dimensional Imaging of Extracellular Matrix and Extracellular

58 4Matrix-Cell Interactions : Current Techniques and Their Limitation s

III. Three-Dimensional Microscopy of Living Systems : Extracellular Matrix and

58 7Extracellular Matrix-Cell Interactions

IV. Summary

59 3References

59 6

28 . Cytometric Analysis of Cell Contact and Adhesio n

Michael S . Kinch

I. Introduction and Application

59 9II. General Strategies to Measure Cell-Cell Adhesions

60 0III. General Strategies to Measure Cell-Ligand Adhesions

60 6IV. Specificity of Cell Adhesion

60 8V. Optimization of Experimental Conditions

60 9References

61 1

29 . Invadopodia : Unique Methods for Measurement of Extracellular Matri x

Degradation in Vitro

Emma T. Bowden, Peter J. Coopman, and Susette C. Muelle r

I. Introduction

61 3II. Invadopodia Activity, a Measurement for Localized Membrane Degradation

61 5III. Fluorescent Activated Cell Sorting-Phagocytosis, a Measurement for

61 9Internalization of Proteolyzed Extracellular Matri x

IV. Protocols

623References

62 6

Index

62 9

Volumes in Series

64 5

Contents of Volume 64Cytometry, Third Edition, Part B

PART VII Cytogenetics and Molecular Genetic s

30 . Sorting of Plant Chromosomes

Jaroslav Dolefel, Martin A . Lysdk, Marie Kubaldkovd, Hana Simkovd, Jiff Macas ,and Sergio Lucretti

31. Quantitative DNA Fiber Mappin g

Heinz-Ulli G . Weier

32. Primed in Situ Labelin g

Johnny Hindkjaer, Lars Bolund, and Steen Kelvra a

33. Measurements of Telomere Length on Individual Chromosomes byImage Cytometry

Steven S . S . Poon and Peter M. Lansdorp

34. Detection of Chromosome Translocation Products in Single Interphas eCell Nuclei

Jingly Fung, Santiago Munne, and Heinz-Ulli G. Weie r

PART VIII Cell Function and Differentiatio n

35. Analysis of Mitochondria by Flow Cytometry

Martin Poot and Robert H. Pierce

36. Analysis of RNA Synthesis by Cytometry

Peter ostrup Jensen, Jacob Larsen, and Jergen K. Larsen

37. Flow Cytometry of Erythropoiesis in Culture : Bivariate Profiles of Fetaland Adult Hemoglobin

Ralph M. Böhmer

38. Flow Cytometric Analysis of Human Hemopoietic Progenito rDifferentiation by Assessing Cell Division Rate and Phenotypic Profil e

Luca Pierelli, Giovanni Scambia, and Andrea Fattorossi

PART IX Experimental Oncology

39. Cytometry of Antitumor Drug-Intracellular Target Interaction s

Paul J. Smith and Marie Wiltshire

40. Monitoring of Cellular Resistance to Cancer Chemotherapy : DrugRetention and Efflux

Awtar Krishan

41. Resistance of Tumor Cells to Chemo- and Radiotherapy Modulated b y

the Three-Dimensional Architecture of Solid Tumors and Spheroid s

Ralph E. Durand and Peggy L . Oliv e

42. Analysis of DNA Damage in Individual Cells

Peggy L . Olive, Ralph E. Durand, Judit P . Bandth, and Peter]. Johnston

43. Cytometric Methods to Analyze Ionizing-Radiation Effect s

William D . Wright, Isabelle Lagroye, Peng Zhang, Robert S . Malyapa ,and Joseph L . Roti Rot i

44. Cytometric Methods to Analyze Thermal Effects

Robert P. VanderWaal, Ryuji Higashikubo, Mai Xu, Douglas R . Spitz ,William D . Wright, and Joseph L. Roti Rot i

PART X Clinical Oncology

45. Multiparameter Data Acquisition and Analysis of Leukocytes by

Flow Cytometry

Carleton C . Stewart and Sigrid J . Stewar t

46. Immunophenotyping of Hematological Malignancies by Lase r

Scanning Cytometry

Richard ]. Clatch

47. Immunophenotyping of Acute Leukemia : Utility of CD45 for BlastCell Identification

J-P. Vial and F . Lacombe

48. Cell Proliferation Markers in Human Solid Tumors : Assessing Their

Impact in Clinical Oncolog y

Maria Grazia Daidone, Aurora Costa, and Rosella Silvestrin i

49. Detection of Minimal Residual Diseas e

Andrzej Deptala and Sharon P . Mayer

50. Analysis of Human Tumors by Laser Scanning Cytanetry

Wojciech Gorczyca, Andrzej Deptala, Elzbieta Bedner, Xun Li, Myron IL Me/awed ,and Zbigniew Darzynkiewicz

51. Laser Cytometry of Human Tissues and Tumors: Proliferation an dTherapeutic Applications

David A . Re w

52. Prediction and Precise Diagnosis of Diseases by Data Pattern Analysis i nMultiparameter Flow Cytometry : Melanoma, Juvenile Asthma, andHuman Immunodeficiency Virus Infection

Günter Valet, Hanna Kahle, Friedrich Otto, Edeltraut Bräutigam, and Luc Kestens

PART XI Microorganisms and Infectious Disease s

53. Flow Cytometric Analysis of Microorganism s

S . A . Sincock and J . Paul Robinson

54. Staining and Measurement of DNA in Bacteri a

Harald B. Stee n

55. Flow Cytometric Monitoring of Bacterial Susceptibility to Antibiotic s

Mette Walberg and Harald B . Steen

56. Flow Cytometry for Evaluation and Investigation of Huma nImmunodeficiency Virus Infection

Thomas W. Mc Closkey