Methods in Cell Biology

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Methods in Cell Biology

VOLUME 41 Flow Cytometr y

SECOND EDITION, PART A

Zbigniew Darzynkiewic z

J. Paul Robinson

Harry A . Crissman

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Contributors

xx i Preface to the Second Edition

xx v Preface to the First Edition

xxi x

1 . Dissociation of Intact Cells from Tumors and Normal Tissue s

Daniel W. Visscher and John D . Crissman I. Introduction

1 II. Application

2 III. Methodology : Mechanical Cell Dispersion

5 IV. Results

6 V. Conclusion

1 0 References

1 3

2 . Assays of Cell Viability : Discrimination of Cells Dyin g by Apoptosis

Zbigniew Darzynkiewicz, Xun Li, and Jianping Gong I. Introduction

1 6 II. Changes in Light Scatter during Cell Death

2 0 III. Cell Sensitivity to Trypsin and DNase

2 0 IV. Fluorescein Diacetate (FDA) Hydrolysis and PI Exclusion

2 1 V. Rh123 Uptake and PI Exclusion

2 2 VI. PI Exclusion Followed by Counterstaining with Hoechst 33342

2 3 VII. Hoechst 33342 Active Uptake and PI Exclusion

2 5 VIII. Controlled Extraction of Low MW DNA from Apoptotic Cells

2 6 IX. Sensitivity of DNAin Situto Denaturation

2 9 X. Detection of DNA Strand Breaks in Apoptotic Cells

29 XI. A Selective Procedure for DNA Extraction from Apoptotic Cell s

Applicable to Gel Electrophoresis and Flow Cytometry

3 1 XII. Comparison of the Methods : Confirmation of the Apoptotic Mod e

of Cell Death

3 6 References

3 7

3 . Cell Preparation for the Identification of Leukocyte s

Carleton C . Stewart and Sigrid J . Stewar t I. Introduction

40 II. Antibodies

40

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III. Titering Antibodies

50 IV. Third Color Reagents

5 1 V. Cell Preparation

5 2 VI. Labeling Procedures

5 4 VII. Lysing Erythrocytes

57 VIII. Detecting Dead Cells

58 IX. Fixation

5 8 X. Solutions and Reagents

5 8 References

5 9

4 . Multiparameter Analysis of Leukocytes by Flow Cytometr y Carleton C . Stewart and Sigrid J . Stewart

I . Introduction

6 1 11 . Correlated List-Mode Data

6 2 Ill . Single-Label Analysis

6 3 IV. Optical Filtration

6 5 V. Compensation

66 VI. Two-Color Fluorescence

7 1 VII. Three-Color Immunophenotyping

73 Vlll . Fixing Cells

7 6 IX. Detecting Dead Cells

7 6 X. Summary

7 8 References

7 8

5 . Immunophenotyping Using Fixed Cell s George F . Babcock and Susan M . Dawes

I. Introduction

8 1 II. Application

8 3 III. Materials

8 3 IV. Cell Preparation and Staining

8 5 V. Critical Aspects of the Procedure

8 6 VI. Results and Discussion

8 7 References

92

6 . Simultaneous DNA Content and Cell-Surfac e Immunofluorescence Analysi s

Paul P .

T.

Brons, Piet E . J . Van Erp, and Arie H . M . Pennings I . Introduction

95 IL Materials and Methods

96 III. Instruments

97 IV. Critical Aspects of the Method

V. Results 9 8

VI. Applications and Future Directions 9 8

References 99

102

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7 . Flow Cytometry Crossmatching for Solid Organ Transplantatio n

Robert A . Bra y I. Introduction

103 II. Applications

104 III. Materials

106 IV. Cell Preparation and Staining

109 V. Critical Aspects of the Procedure

11 1 VI. Standards

11 4 VII. Instruments

11 5 VIII. Results and Discussion

11 6 References

11 8

8 . Cell Membrane Potential Analysi s

Howard M. Shapiro I. Introduction

12 1 II. Materials and Methods

12 5 III. Improving Cytometry of Membrane Potentials

130 References

133

9 . Measurement of Intracellular p H

Michael J . Boyer and David W . Hedley I. Introduction

135 II. Application

140 III. Materials

140 IV. Cell Preparation and Staining : BCECF or SNARF

14 1 V. Instruments

14 2 VI. Critical Aspects

144 VII. Results and Discussion

147 References

14 8

10. Intracellular Ionized Calciu m

Carl H . June and Peter S . Rabinovitch I. Introduction

15 0 II. Flow Cytometric Assay With Indo-1

15 2 III. Use of Flow Cytometry and Fluo-3 to Measure [Ca z± ] ;

16 1 IV. Use of Flow Cytometry and Fluo-3/Fura-Red for Ratiometri c

Analysis of [Caz} ] ;

16 3 V. Cell Conjugate Assays Combined with Calcium Analysis and

Flow Cytometry

16 5 VI. Pitfalls and Critical Aspects

16 5 VII. Limitations

170 VIII. Results

172 References

172

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11 . Cellular Protein Content Measurement s Harry A . Crissman and John A . Steinkam p

I. Introduction

17 5 II. Applications

17 6 III. Materials

179 VI. Controls and Standards

179 VII. Instruments

180 VIII. Results and Discussion

18 1 References

18 3

12 . Lysosomal Proton Pump Activity : Supravital Cell Staining wit h Acridine Orange Differentiates Leukocyte Subpopulation s Frank Traganos and Zbigniew Darzynkiewic z

I. Introduction

186 III. Materials

18 7 IV. Staining Procedures

18 8 VI. Controls and Standards

190 VIII. Results

19 1 References

19 4

13 . Staining of DNA in Live and Fixed Cell s Harry A . Crissman and Gregory T . Hirons

I. Introduction

196 II. Applications with DNA-Specific Fluorochromes

197 III. Materials

20 2 IV. Cell Preparation and Fixation

20 2 V. Cell Staining

203 VI. Critical Aspects

204 VII. Controls and Standards

20 5 Vlll . Instruments

20 5 IX . Results and Discussion

20 5 References

20 8 14 . High-Resolution Analysis of Nuclear DNA Employing th e

Fluorochrome DAPI Friedrich J . Otto

I. Introduction

II. Applications 21 1

III. Materials 21 2

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IV. Cell Separation and Fixation Method

21 3 V. Staining Procedure

21 4 VI. Instrument Setup

21 4 VII. Standards

21 4 VIII. Results and Discussion

21 5 References

21 7

15 . Detergent and Proteolytic Enzyme-Based Techniques for Nuclea r Isolation and DNA Content Analysi s

Lars L . Vindelev and Lb Jade Christensen I. Introduction

21 9 II. Basic Principles of the Methods

22 1 III. Applications

222 IV. Materials

222 V. Methods

224 VI. Results

227 References

229

16 . DNA Analysis from Paraffin-Embedded Block s David W . Hedley

I. Introduction

232 II. Application

23 2 III. Methods

232 IV. Critical Aspects of Technique

235 V. Alternative Methods for Sample Preparation

23 8 References

240

17 . Controls, Standards, and Histogram Interpretation in DN A Flow Cytometry

Lynn G . Dressler and Larry C . Seame r I. Introduction

242 II. Application

244 III. Instrument Quality Control and Standardization

24 9 IV. DNA Histogram Interpretation

25 3 V. Methods

25 6 VI. Summary of Criteria for Interpretation of DNA Histograms

25 9 References

26 1

18 . DNA Content Histogram and Cell-Cycle Analysi s Peter S . Rabinovitch

I . Introduction

264 It . DNA Content Histogram Basic Principles

264 III. Cell-Cycle Analysis of DNA Content Histograms

266 IV. Critical Aspects of DNA Content and Cell-Cycle Analysis

269

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V. Interpretation of DNA Content and Cell-Cycle Histograms

28 5 VI. Conclusion

29 3

References

29 4

19 . Immunochemical Quantitation of Bromodeoxyuridine : Application to Cell-Cycle Kinetic s

Frank Dolheare and Jules R . Selde n I. Introduction

29 8 III. Materials

29 9 IV. Procedures

3U 3 V. Critical Aspects of the Procedure

3 11 7 VI. Controls and Standards

3 11 9 Vll . Instruments

3 11 1 VIII. Results

311 1 IX. Summary

31 3

References

31 5

20 . Application and Detection of IdUrd and C1dUrd as Tw o Independent Cell-Cycle Marker s

Jacob A . Aten, Jan Stop, Ron Hoebe, and Piet J . M . Bakke r I. Introduction

31 7 II. Application

31 8 III. Materials

31 9 IV. Methods

32 3 VII. Results

32 4

References

325

21 . Cell-Cycle Analysis Using Continuous Bromodeoxyuridin e Labeling and Hoechst 33358-Ethidium Bromide Bivariat e Flow Cytometry

Martin Poor, Hofer Hoehn, Manfred Kubbies, Angelika Grossmann , Yuchyau Chen, and Peter S . Rabinovitd i

I. Introduction

II. Application 32 8

111 . Materials 328

IV. Procedures 329

V. Controls and Standards 33 1

VI. Instruments 33 3

VII. Results 33 3

33 4

References

339

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22 . Detection of BrdUrd-Labeled Cells by Differential Fluorescenc e Analysis of DNA Fluorochromes : Pulse-Chase and Continuou s Labeling Methods

Harry A . Crissman, Noboru Oishi, and Robert Habberset t I. Introduction

34 3 III. Materials

34 4 IV. Cell Preparation and Staining Procedure

34 4 V. Critical Aspects

34 5 VIII. Results

346 References

349 23 . Analysis of Intracellular Protein s

Kenneth D . Bauer and James W. Jacobberge r I. Introduction

35 6 III. Materials

35 9 IV. Staining Procedures

36 1 V. Critical Aspects

370 VIII. Multiparameter Analysis of Intracellular Antigens

37 1 References

374

24 . "Washless" Procedures for Nuclear Antigen Detectio n Jergen K . Larsen

I. Introduction

37 8 III. Materials, Cell Preparation, and Staining

38 U IV. Critical Aspects of the Procedure : Controls, Standards, and

Instruments

38 2 V. Results

38 7 References

38 8

25 . Light Scatter of Isolated Cell Nuclei as a Parameter Discriminatin g the Cell-Cycle Subcompartment s

Walter Giaretti and Michael Niisse I. Introduction

39 1 III. Materials

39 1 IV. Critical Aspects of the Procedure

393

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V. Instruments

39 4 VI. Results

39 5 VII. Appendix

39 8

References

400

26 . Simultaneous Analysis of Cellular RNA and DNA Conten t Zbigniew Darzynkiewic z

I. Introduction

4116 III. Materials

407 IV. Staining Procedures Employing AO

409 V. Staining RNA and DNA with PY and Hoechst 33342

41 0 VI. Critical Aspects of the Procedures

41 1 VII. Controls and Standards

41 3 VIII. Instruments

41 4 IX. Results

41 5 X. Comparison of the Methods

41 8

References

41 9

27 . Analysis of DNA Content and Cyclin Protein Expression i n Studies of DNA Ploidy, Growth Fraction, Lymphocyt e Stimulation, and the Cell Cycl e

Zbignietv Darzynkiewicz, Jianping Gong, and Frank Traaano s I. Introduction

42 2 II. Bivariate Analysis of DNA Content versus Cyclin B 1

or Cyclin E Expression

422 III. Application of Cyclin B1 Analysis in DNA Ploidy and Chromati n

Cycle Estimates

424 IV. Lymphocyte Stimulation

42 7 V. Tumor Growth Fraction Analysis

42 9 VI. Mapping the Cell Cycle : Mechanism of Action of Cytostatic Drugs

43 1 VII. Critical Points of the Procedure

VIII. Conclusions 433

434

References

43 5

28 . Oxidative Product Formation Analysis by Flow Cytometr y J . Paul Robinson, Wayne O . Carter, and Padma Kumar Narayana n

I . Introduction

q . Oxidative Burst 437

III. Phagocytosis 43 8

IV. Controls and Standards 442

44 7

References

447

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29 . Flow Cytometric Determination of Cysteine and Serine Proteinase Activities in Living Cells with Rhodamine 110 Substrate s

Sven Klingel, Gregor Rothe, Wolfgang Kellermann, and Günter Valet I. Introduction

45 0 III. Results

45 4 IV. Discussion

45 7 References

45 8

30 . Leucine Aminopeptidase Activity by Flow Cytometr y John]. Turek and J . Paul Robinso n

I. Introduction

462 III. Materials

46 2 IV. Methods

463 VI. Controls

46 4 VIII. Results

46 4 References

46 6

31 . Enzyme Kinetic s

James V. Watson and Caroline Div e I. Introduction

46 9 II. Classical Enzyme Kinetics

47 0 III. Incorporation of Time

47 5 IV. Flow Cytoenzymology

477 Appendix 1

503 Appendix 2

50 6 References

506

32 . On-Line Flow Cytometry : A Versatile Method fo r Kinetic Measurement s

Kees Nooter, Hans Herweijer, Richard R . Jonker, and Geri. van den Engh I. Introduction

50 9 II. Instrumentation

51 0 III. Applications

51 6 References

525

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33 . Acid-Induced Denaturation of DNA in ^Situ as a Probe

o f

Chromatin Structur e

Zhigniew Darzynkiewirz 1 . Introduction

527 II. Application

530 III. Materials

53 3 VI. Standards

535 Vll . Instruments

536 V III. Results

537 References

54 1

34 . Detection of Intracellular Virus and Viral Product s Judith La(iu and John M . Lehma n

I. Introduction

546 III. Critical Aspects of the Procedure

549 IV. Flow Analysis

55 3 V. Applications

55 4 References

55 7

Index

55 9 Volumes in Series

587

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33 . Acid-Induced Denaturation of DNA in Situ as a Probe of Chromatin Structure

Zbi,i view Darzynkiewir z 1 . Introduction

52 7 II . Application

53 0 Ill . Materials

53 3 VI. Standards

535 Vll . Instruments

536 V III. Results

53 7 References

54 1

34 . Detection of Intracellular Virus and Viral Product s Judith Lai/in and John M . Lehman

I. Introduction

546 III. Critical Aspects of the Procedure

549 IV. Flow Analysis

55 3 V. Applications

55 4 References

55 7

Index

55 9 Volumes in Series

587