Methods in Cell Biology

VOLUME 64CytometryThird Edition, Part B

Zbigniew Darzynkiewicz

Harry A. Crissman

J. Paul Robinson

Contents of Volume 63

xi i

Contributors

x v

Preface to the Third Edition

xix

Preface to the Second Edition

xxiii

Preface to the First Edition

xxvi i

PART VII Cytogenetics and Molecular Genetics

30 . Sorting of Plant Chromosomes

Jaroslav Doleel, Martin A . Lysdk, Marie Kubaldkovd, Hana Simkovit, Jiff Mat-as ,and Sergio Lucretti

I. Introduction

3

II. Application

5

III. Materials

6

IV. Procedures

1 2

V. Critical Aspects of the Procedures

1 8

VI. Instruments

2 1

VII. Results

2 1

VIII. Conclusions and Perspectives

2 6

References

2 8

31 . Quantitative DNA Fiber Mapping

Heinz-Ulli G . Weier

I. Introduction

3 3

II. Materials

3 5

III. Protocols

3 8

IV. Critical Aspects of the Procedure

4 6

V. Results and Discussion

4 7

References

5 2

32 . Primed in Situ Labelin g

Johnny Hindkjaer, Lars Bolund, and Steen Kelvra a

I. Introduction

5 5II. Applications

5 6

III. Materials

5 6

IV. Protocols

58

V. Critical Aspects of the Procedures

66

References

67

33 . Measurements of Telomere Length on Individual Chromosomes b y

Image Cytometry

Steven S. S . Poon and Peter M. Lansdorp

I. Introduction

7 0

II. Background

7 2

III. Methods

7 7

IV. Results

88

V. Discussion

92References

94

34 . Detection of Chromosome Translocation Products in Single Interphas e

Cell Nucle i

Jingly Fung, Santiago Munne, and Heinz-Ulli G . Weier

I. Introduction

9 8II. Materials

9 9

III. Protocols

10 2

IV. Results and Discussion

108

V. Critical Aspects of the Procedure

11 1

VI. Applications

11 2

References

11 3

PART VIII Cell Function and Differentiatio n

35 . Analysis of Mitochondria by Flow Cytometry

Martin Poot and Robert H . Pierce

I. Introduction

11 8

II. Materials and Methods

12 1

III. Critical Aspects

12 5

References

12 6

36 . Analysis of RNA Synthesis by Cytometry

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

I. Introduction

12 9

II. Background

13 0

III. Methods for Analysis of 5'-Bromouridine Incorporation and

13 1

DNA Conten t

IV. Results of Labeling RNA with 5 ' -Bromouridine

134

V. Applications

13 6

References

137

37 . Flow Cytometry of Erythropoiesis in Culture : Bivariate Profiles of Feta l

and Adult Hemoglobin

Ralph M. Böhmer

I. Introduction

13 9II. Details of the Method

14 1III. Sample Experiments

143IV. Problems and Limitations

14 9References

15 1

38 . Flow Cytometric Analysis of Human Hemopoietic Progenito r

Differentiation by Assessing Cell Division Rate and Phenotypic Profil e

Luca Pierelli, Giovanni Scambia, and Andrea Fattoross i

I. Introduction

15 3II. Background

15 5III. Critical Aspects of Methodology

15 6IV. Functionally Distinct Circulating Hemopoietic Progenitor Subsets Can Be

15 7Assessed during Cytokine-Driven Differentiatio n

V. Concluding Remarks

16 7References

16 9

PART IX Experimental Oncology

39 . Cytometry of Antitumor Drug-Intracellular Target Interactions

Paul J. Smith and Marie Wiltshire

I. Introduction

17 3II. General Classification of Cytotoxic Anticancer Agents

17 4III. Establishment of Quality Control Parameters

17 8IV. Drug-DNA Interactions

18 3V. Conclusions

18 8References

19 0

40 . Monitoring of Cellular Resistance to Cancer Chemotherapy: Dru g

Retention and Efflux

Awtar Krisha n

I. Introduction and Background

19 3II. Applications

19 6III. Cell Lines, Efflux, Multiple Drug Resistance Drugs, and Blockers

19 6IV. Staining Protocols

19 7V. Critical Aspects

19 8VI. Controls, Standards, and Instruments

19 9VII. Results and Conclusions

20 1References

206

41 . Resistance of Tumor Cells to Chemo- and Radiotherapy Modulated b ythe Three-Dimensional Architecture of Solid Tumors and Spheroid s

Ralph E . Durand and Peggy L. Olive

I. Introduction and Historical Perspective

21 1

II. Background

21 3

III. Methods

21 5

IV. Results and Discussion

21 7

V. Conclusions and Future Directions

22 7

References

22 8

42 . Analysis of DNA Damage in Individual Cell s

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

I. Introduction

23 5

II. The Development of the Single Cell Gel Electrophoresis/Comet Assay

23 6

III. Comet Preparation and Analysis

23 8

IV. Types of Damage Detected by the Comet Assay

242

V. Using DNA Damage to Predict Cell Survival in Complex Systems

244

VI. Future Directions

246

References

247

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

I. Introduction

25 1

II. Applications

252

III. Methods for Measuring DNA Damage on a Cell-by-Cell Basis

253

IV. Summary

267

References

267

44 . Cytometric Methods to Analyze Thermal Effect s

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

I. Introduction

26 9

II. Application

27 0

III. Methods to Measure Nuclear and Nuclear Matrix Protein Content

27 1

IV. Identification of Altered DNA Replication Patterns Following Heat Shock

27 6

V. Nuclear Localization of hsp70

27 8

VI. Prooxidant Measurement

28 0

VII. Results

28 1

References

285

PART X Clinical Oncology

45 . Multiparameter Data Acquisition and Analysis of Leukocytes by

Flow Cytometry

Carleton C . Stewart and Sigrid' . Stewart

I. Introduction

28 9II. Correlated List Mode Data

29 0III. Verification of Instrument Performance

29 3IV. Optical Filtration and Spectral Compensation

29 5V. Multiparameter Data Analysis

30 8VI. Summary

31 1References

31 1

46 . Immunophenotyping of Hematological Malignancies by Lase r

Scanning Cytometry

Richard J. Clatch

I. Immunophenotyping of Leukemia and Lymphoma: General Considerations

31 3II. Description of Laser Scanning Cytometry

31 6III. Immunophenotyping by Laser Scanning Cytometry

31 6IV. Extensions of the Method

32 7V. Conclusions

34 0References

34 0

47 . Immunophenotyping of Acute Leukemia : Utility of CD45 for Blas t

Cell Identification

J-P. Vial and F . Lacombe

I. Introduction

344II. Background

344III. Methods

34 5IV. Results and Comparison with Other Methods

34 8V. Critical Aspects of the Methodology

35 2VI. Pitfalls and Misinterpretation of the Data

35 3VII. Future Directions

35 5References

35 7

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

Impact in Clinical Oncology

Maria Grazia Daidone, Aurora Costa, and Rosella Silvestrin i

I. Introduction

35 9II. Proliferation Markers

36 1III. Biological Studies

365IV. Clinical Studies

36 6V. Conclusions on First Generation Translational Studies with

37 7Proliferation MarkersReferences

378

49 . Detection of Minimal Residual Diseas e

Andrzej Deptala and Sharon P . Mayer

I . Introduction

38 5IL Tissue Sources to Detect Minimal Residual Disease

38 7III. Methods to Detect Minimal Residual Disease

38 8IV. Technical Problems

41 4V. Concluding Remarks

41 6

References

41 6

50 . Analysis of Human Tumors by Laser Scanning Cytometr y

Wojciech Gorczyca, Andrzej Deptala, Elzbieta Bedner, Xun Li, Myron R. Melarned,

and Zbigniew Darzynkiewicz

I . Introduction

42 2

11 . Analysis of Cellular DNA Content by Laser Scanning Cytometry

423

III. Analysis of Apoptosis by Laser Scanning Cytometry

425

IV. Analysis of Proliferation Associated Antigens by Laser Scanning Cytometry

43 0

V. Analysis of Estrogen Receptors by Laser Scanning Cytometry

43 5

VI. Analysis of Transcription Factors by Laser Scanning Cytometry

437

VII. Measurement of Nucleoli Using Laser Scanning Cytometry Fluorescence in

43 8

Situ Hybridization ProtocolReferences

44 1

51 . Laser Cytometry of Human Tissues and Tumors : Proliferation and

Therapeutic Applications

David A . Rew

I. Introduction

44 6

II. Ploidy and Proliferation in Surgical Oncology

447

III. Cytometric Studies of Proliferation

44 8IV. The Halogenated Pyrimidines in Cell Proliferation Research

44 9

V. Clinical Studies of Cell Production Rates with Thymidine Analogs

45 2

VI. Further Applications of Cytometry in Clinical Oncology

47 4

VII. Conclusions

47 8

References

47 8

52 . Prediction and Precise Diagnosis of Diseases by Data Pattern Analysis i n

Multiparameter Flow Cytometry: Melanoma, Juvenile Asthma, and

Human Immunodeficiency Virus Infection

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

I. Introduction

48 8

II. Material and Methods

48 9

III. Results

49 3

IV. Discussion

50 6References

507

PART XI Microorganisms and Infectious Disease s

53 . Flow Cytometric Analysis of Microorganism s

S . A . Sincock and J. Paul Robinso n

I. Introduction

51 1II. Experimental Approaches

51 4III. Applications in Medical and Food Microbiology

52 6IV. Conclusion

53 1

References

53 2

54 . Staining and Measurement of DNA in Bacteri a

Harald B . Steen

I. Introduction

53 9II. Basic Considerations

54 0III. Experimental Methods

54 3IV. Standards and Controls

54 8

References

55 0

55 . Flow Cytometric Monitoring of Bacterial Susceptibility to Antibiotic s

Mette Walberg and Harald B . Stee n

I. Introduction

553II. Fluorescent Dyes

55 4III. Uptake of Fluorescent Dyes

55 5IV. Effects of Antibiotics

55 8V. Assessment of Drug Effects

55 8VI. Applications of Flow Cytometry to Medical Microbiology

563

References

563

56 . Flow Cytometry for Evaluation and Investigation of Human

Immunodeficiency Virus Infectio n

Thomas W. Mc Closkey

I. Introduction

56 7II. Application of Flow Cytometry to Monitor HIV Infection

56 8III. Application of Flow Cytometry to Investigate HIV Disease

58 2IV. Conclusions

58 4

References

58 4

Index

593

Volumes in Series

609

Contents of Volume 63Cytometry, Third Edition, Part A

PART I Principles of Cytometry and General Method s

1. A Brief History of Flow Cytometry and Sortin g

Myron R . Melame d

2. Principles of Flow Cytometry : An Overvie w

Alice L. Giva n

3. Laser Scanning Cytometry

Louis A . Kamentsk y

4. Principles of Confocal Microscopy

J. Paul Robinson

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

Absorption, and Fluorescence

Howard M. Shapiro

6. Flow Cytometric Fluorescence Lifetime Measurements

Harry A . Crissman and John A . Steinkamp

7. Principles of Data Acquisition and Display

Howard M . Shapiro

8. Time as a Flow Cytometric Parameter

Larry Seamer and Larry A . Sklar

9. Protein Labeling with Fluorescent Probe s

Kevin L. Holmes and Larry M. Lantz

PART II Cell Preparation

10. Preparation of Cells from Bloo d

J. Philip McCoy, Jr .

11. Cell Preparation for the Identification of Leukocyte s

Carleton C . Stewart and Sigrid J. Stewart

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

Steven K. Koester and Wade E. Bolto n

PART III Standardization, Quality Assuranc e

13. Stoichiometry of Immunocytochemical Staining Reaction s

James IN. Jacobberger

14. Standardization and Quantitation in Flow Cytometr yRobert A . Hoffman

PART IV Cell Proliferation

15. Methods to Identify Mitotic Cells by Flow Cytometry

Gloria Juan, Frank Traganos, and Zbigniew Darzynkiewicz

16. Cell Cycle Kinetics Estimated by Analysis o fBromodeoxyuridine Incorporation

Nicholas H. A . Terry and R . Allen White

17. Flow Cytometric Analysis of Cell Division History Using Dilution o fCarboxyfluorescein Diacetate Succinimidyl Ester, a Stably Integrate dFluorescent Prob e

A. Bruce Lyons, Jhagvaral Hasbold, and Philip D . Hodgkin

18. Antibodies against the Ki-67 Protein : Assessment of the Growth Fractionand Tools for Cell Cycle Analysi s

Elmar Endl, Christiane Hollmann, and Johannes Gerdes

19. Detection of Proliferating Cell Nuclear Antigen

Jargen K. Larsen, Göran Landberg, and Göran Roos

20. Lymphocyte Activation Associated Antigens

Andrea Fattorossi, Alessandra Battaglia, and Cristiano Ferlini

PART V Cell Death/Apoptosi s

21. Analysis of Mitochondria during Cell Deat h

Andrea Cossarizza and Stefano Salviol i

22. Cytometry of Caspases

Steven K. Koester and Wade E . Bolto n

23. Analysis of Apoptosis in Plant Cells

Iona E . Wei r

24. Difficulties and Pitfalls in Analysis of Apoptosi s

Zbigniew Darzynkiewicz, Elzbieta Bedner, and Frank Tragano s

PART VI Cell-Cell, Cell-Environment Interactions

25. Analysis of Cell Migratio n

Nicole Dodge Zantek and Michael S . Kinch

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

Sherry L . Voytik-Harbin

27. Three-Dimensional Imaging of Extracellular Matrix and Extracellular

Matrix-Cell Interaction s

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

28. Cytometric Analysis of Cell Contact and Adhesio n

Michael S . Kinch

29. Invadopodia : Unique Methods for Measurement of Extracellular Matrix

Degradation in Vitro

Emma T. Bowden, Peter J. Coopman, and Susette C . Mueller