Methods in Cell Biology

VOLUME 75

Cytometry, 4th Edition: New Developments

Zbigniew Darzynkiewicz

Mario Roederer

Hans Tanke

Contributors

xvi i

Preface

xxii i

Preface to the Third Edition

xxvi i

Preface to the Second Edition

xxx i

Preface to the First Edition

xxxv

PART I Instrumentation/Fluorochrome s

1 . Optimization of Emission Optics for Multicolor Flow Cytometry

Nicole Baumgarth and Marty Bigo s

I. Introduction

3II. Fluorochromes

4III. Hardware Components Affecting Light Collection

8IV. Maximizing Signal Intensities

1 1V. Maximizing Signal Collection Quality

1 1VI. Emission Collection Optimization

1 4VII. Testing Emission Optics

1 6References

2 1

2 . Two-Photon Tissue Cytometry

Timothy Ragan, Ki Hean Kim, Karsten Bahlmann, and Peter T . C . So

I. Introduction

2 3II. Technical Development of Two-Photon Tissue Cytometry

2 4III. Experimental Demonstration of Two-Photon Tissue Cytometry

3 2IV. Future Outlook

3 7V. Conclusion

3 7References

38

3 . Confocal Microscopy : Quantitative Analytical Capabilities

Jurek W. Dobruck i

I. Introduction

4 2

II. Cartesian Coordinates, Distance, Size, Shape, and Volume in

Confocal Microscopy

4 3

III. Measurements of Fluorescence Intensity in Confocal Microscopy

5 3

IV. Time Resolution in Confocal Microscopy

5 7

V. Confocal Techniques Based on Measurements of Fluorescenc e

Intensity vs Time

5 7

VI. Spectrally Resolved Confocal Microscopy

6 4

VII. Conclusion

6 9

References

7 0

4 . Surface-Plasmon-Coupled Emission: New Technology for Studying

Molecular Processe s

Zygmunt Gryczynski, Ignacy Gryczynski, Evgenia Matveeva, Joanna Malicka ,

Kazimierz Nowaczyk, and Joseph R . Lakowicz

I. Introduction

7 3

II. Overview of Surface Plasmon Resonance

7 5

III. Surface-Plasmon-Coupled Emission

8 0

IV. Experimental Studies of Surface-Plasmon-Coupled Emission

8 3

V. Practical Applications

9 0

VI. Conclusion

10 2

References

10 2

5 . Cytometry of Fluorescence Resonance Energy Transfe r

György Vereb, Jdnos Matkö, and _linos Szöllösi

I. Introduction

105

II. Theory of FRET

107

III. Measuring FRET

109

IV. Applications

132

V. Perspectives

142

VI. Conclusions

14 5

References

14 6

6 . The Rainbow of Fluorescent Proteins

David W. Galbrait h

I. The Fluorescent Proteins

15 3

II. Flow Analysis Using Fluorescent Proteins

159

III. Flow Sorting Using Fluorescent Proteins

163IV. Conclusion

164

References

165

7. Labeling Cellular Targets with Semiconductor Quantum Dot Conjugate s

Xingyong Wu and Marcel P . Bruchez

I. Introduction

17 1

II. Selection of QDs and Their Conjugates

172III. Labeling of Fixed Cells for Fluorescence Microscopy

173

IV. Measurements on Living Cells Using QD Conjugates

179V. Conclusion

18 1

References

182

8. Next-Generation Laser Scanning Cytometr y

Ed Luther, Louis Kamentsky, Melvin Henriksen, and Elena Holde n

I. Introduction

186II. LSC-Hybrid Technology

186III. Description of the LSC Platform

188IV. Selected Applications

203V. Utility of Solid-Phase Imaging Cytometry in Life Science Research

and Drug Discovery

21 7

References

21 7

PART II General Technique s

9. Biohazard Sorting

Ingrid Schmid, Mario Roederer, Richard A . Koup, David Ambrozak ,and Stephen P. Peletto

I. Introduction

22 1II. Critical Aspects of the Procedure

222III. Applications and Future Directions

23 8

References

23 9

10. Guidelines for the Presentation of Flow Cytometric Data

Mario Roederer, Zbigniew Darzynkiewicz, and David R . Parks

I. Introduction

241

II. General Principles of Graphical Presentation

243

III. Statistics

25 1IV. Subset Analysis

25 3

V. Conclusion

25 6

References

25 6

11 . Mechanism of Antitumor Drug Action Assessed by Cytometr y

Frank Tragano s

I. Introduction

25 8

II. Choice of a Model System

258

III. Drug Dose and Length of Exposure

263

IV. Single-Parameter DNA Histogram Analysis

26 5V. Multiparameter Approaches

27 4

VI. Data Presentation

29 8

VII. Summary and Future Directions

29 9

References

30 1

12 . Cytometric Methods to Detect Apoptosi s

Zbigniew Darzynkiewicz, Xuan Huang, Masaki Okafuji, and Malcolm A . King

I . Introduction

30 8

It . Physical and Molecular Features of Cells Dying by Apoptosis or Necrosis

31 0

Ill . Different Time-Windows for Detection of Apoptosis : Possible Source

of Error in Measurement of Incidence of Apoptosis

31 6

IV. Light-Scattering Properties of Cells Dying by Apoptosis

31 7

V. Mitochondrial Trausmembrane Potential ( P,, 1 )

31 8

VI. Activation of Caspases

32 1

VII. Annexin V Binding

325

VIII. DNA Fragmentation

326

IX. Susceptibility of DNA to Denaturation

32 9

X. Activation of " Tissue " Transglutaminase

33 1

XI. Measuring Incidence of Apoptosis : Which Method to Choose?

33 2

References

33 5

13 . Real-Time Analysis of Apoptosis In Vivo

Pui Lee and Mark S . Sega l

I. Introduction

34 3

II. In Vivo Membrane Markers

34 4

III. A New Generation of Caspase Markers

34 8

IV. Conclusion

35 1

References

351

14 . Detection of DNA Damage in Individual Cells by Analysis of Histon eH2AX Phosphorylation

Peggy L . Olive

I. Introduction

35 5II. Considerations in the Use of 7H2AX as a Measure o f

Double-Stranded Breaks

35 6III. Methods of Analysis

359IV. Typical Results

363V. Possible Applications

370VI. Conclusion

37 1References

37 1

15 . Cytometry of Freshwater Phytoplankto n

Jörg Toepel, Christian Wilhelm, Armin Meister, Annette Becker,and Maria del Carmen Martinez-Ballest a

I. Introduction

37 6II. Background

37 8III. Methods

387IV. Results

393V. Limitations and Applications

399VI. Future Directions

403References

403

16 . Multiplexed Microsphere Assays for Protein and DNA Binding Reaction s

Kathryn L . Kellar and Kerry G . Oliver

I. Introduction

41 0II. Technology and Instrumentation

41 1III. Methods

41 5IV. Results

41 8V. Software

420VI. Critical Aspects of the Methodology

42 1VII. Comparison with Other Methods

424VIII. Future Directions

42 5References

426

PART III Immunology/T-Cell Response s

17. Flow Cytometry Applications of MHC Tetramers

John D . Altma n

I. Introduction

433II. Materials and Methods

436III. Analyzing Tetramer Data

444IV. Combining Tetramer Staining with Functional Assays

446V. Conclusion

447References

448

18 . Use of Peptides and Peptide Libraries as T-Cell Stimulants in Flow Cytometric Studie s

Georgy Cherepnev, Hans-Dieter Volk, and Florian Ker n

I. Introduction

45 4II. Background

455III. Results/Discussion of the Literature

45 9

IV. Technical Considerations

464

V. Specific Applications

467VI. Potential Clinical Use

47 1VII. Protocols

473References

475

19 . Flow Cytometric Analysis of Human Antigen-Specific T-Cell Proliferatio n

Jason M. Brenchley and Daniel C . Doue k

I. Introduction and Background

48 2II. Methods

48 3III. Results

48 4IV. Critical Aspects of the Methodology

48 7V. Pitfalls and Misinterpretation of the Data

49 0VI. Future Directions

491References

49 2

20 . Detection of T-Cell Degranulation : CD107a and b

Michael R . Betts and Richard A . Koup

I. Introduction : Methods to Assess CD8+ T-Cell Function

49 7II. Materials and Methods

500III. Results

504

IV. Critical Aspects

50 5

V. Applications

50 8

References

51 0

21 . T-Cell Responses to Cance r

Peter P. Lee

I. Introduction

51 4II. Methods to Identify and Enumerate TAA-Specific T Cells

51 6

III. Important Considerations

52 0

IV. Methods to Further Analyze In Vivo Biology of Antitumor T Cells

522

V. Novel Issues and Methods

524

VI. Conclusion

527

References

528

PART IV Multi-Color Immunophenotypin g

22 . Multicolor Flow Cytometric Analysis in SIV-Infected Rhesus Macaqu e

Joshua M. Walker, Holden T. Maecker, Vernon C . Maino, and Louis J. Picke r

I. Introduction

536

II. Methods

537

III. Flow Cytometry Instrumentation and Software

53 8

IV. Instrument Setup

539

V. Critical Aspects of the Technology

542

VI. Application of 10-Color Analysis to SIV Infection

54 8

VII. Conclusion

554

References

55 5

23 . Multicolor Immunophenotyping : Human Immune System Hematopoiesis

Brent Wood

I. Introduction

55 9

II. Methodology

56 0

III. Normal Immunophenotypic Patterns of Maturation

56 1

IV. Abnormal Immunophenotypic Patterns of Maturation

57 3

References

575

24 . Multicolor Immunophenotyping : Human Mature Immune Syste m

Stephen C . De Rosa

I. Introduction

57 7

II. Choice of Markers

57 8

III. Staining Panels : Matching Fluorochrome with Cell Marker

58 6

IV. Conclusion

59 2

References

59 3

25 . Differential Diagnosis of T-Cell Lymphoproliferative Disorders by Flow Cytometr y

Multicolor Immunophenotyping. Correlation with Morphology

Wojciech Gorczyca

I. Introduction

59 6

II. Materials

59 6III. Methods

597

IV. Identification of Abnormal T-Cell Population by Flow Cytometry

59 8V. Precursor T Lymphoblastic Lymphoma/Leukemia

60 1

VI. Peripheral (Mature/Post-thymic) Lymphoma Versus Precurso rT-Lymphoblastic Lymphoma/Leukemia

60 1

VII. Thymocytes from Thymic Hyperplasia/Thymoma Versus Precurso r

T-Lymphoblastic Lymphoma/Leukemia

60 4

VIII. Mature T-Cell Lymphoproliferative Disorders

60 4

IX. CD117 Expression in T-Cell Lymphoproliferations

61 6

X. Blastic NK Cell Lymphoma (DC-2 Acute Leukemia)

61 8

XI. Conclusion

61 8

References

61 9

26 . Isolation and Immunophenotyping of Human and Rhesus Macaqu e

Dendritic Cells

Karin Lore

I. Introduction

62 3

II. In Vitro Differentiation of Monocyte-Derived Dendritic Cells

62 5

III. Detection and Sorting Procedures of Subsets of Dendritic Cells

62 5

IV. Culture and Activation of Dendritic Cells

63 5

References

63 7

27 . B-Cell Immunophenotypin g

Nicole Baumgarth

I . Introduction

643

11 . Immunophenotyping of B-Cell Developmental Stages in

the Bone Marrow

644III. Peripheral B-Cell Populations

649IV. Antigen-Induced B-Cell Subsets

65 5V. Antigen-Specific B Cells

65 8References

660

PART V Other Topics in Immunology

28 . Plow Cytometry Inununophenotypic Characteristics of Monocytic Populatio nin Acute Monocytic Leukemia (AML-M5), Acute Myelomonocytic Leukemi a(AML-M4), and Chronic Myelomonocytic Leukemia (CMML)

Wojciech Gorczyea

I. Introduction

66 6II. Materials

66 6III. Methods

667IV. Acute Monocytic Leukemia

668V. Chronic Myelomonocytic Leukemia

668VI. Acute Myelomonocytic Leukemia

66 8VII. Differential Diagnosis

670VIII. Conclusion

676References

676

29 . Phagocyte Functio n

Gregor Rothe and Mariam Klouch e

I. Introduction

680II. Background

690III. Methods

694IV. Results

697V. Pitfalls and Misinterpretation of the Data

69 8VI. Comparison with Other Methods

700VII. Applications and Biomedical Information

70 1VIII. Future Directions

703References

703

30 . Neutralizing Antibody Quantification by Flow Cytometry

John R . Mascola

I . Introduction

709

II. Background

71 0

III. Results

71 2IV. Discussion and Key Points

71 4

References

71 5

PART VI Cytogenetics

31 . Telomere Length Measurements Using Fluorescence In Situ Hybridizatio nand Flow Cytometry

Gabriela M. Baerlocher and Peter M . Lansdorp

I. Introduction

72 0II. Background

72 5

III. Methods

72 7

IV. Results

73 9V. Critical Aspects of Methodology

74 2

VI. Pitfalls and Misinterpretation of Data

74 4

VII. Comparison with Other Methods

74 5

VIII. Applications

74 6

IX. Future Directions

74 8

References

74 9

32 . Detecting Copy Number Changes in Genomic DNA : MAPH and MLPA

Stefan J . White, Martgn H . Breuning, and Johan T. den Dunnen

I. Introduction

75 1

II. Reactions

75 2

III. Analysis

75 6

IV. Applications

76 2

V. Future Possibilities

76 4

References

76 6

33 . Genomic Array Technology

Heike Fiegler and Nigel P. Carter

I. Introduction

76 9

II. Construction of Genomic Clone Arrays Using DOP-PCR Amplification

77 0

III. Validation of Array Performance and Data Analysis

77 1

IV. Array CGH

77 6

V. Array CGH for Cytogenetic Analyses

77 8

VI. Array Painting

779

VII. Application of Array CGH and Array Painting for Complet eCytogenetic Analyses

780VIII. ChIP on Genomic Clone Arrays

782IX. Conclusion

782References

784

34 . Prospects for In Situ Analyses of Individual and Complexes of DNA, RNA, and Protei n

Molecules with Padlock and Proximity Probes

Ulf Landegren, Mats Nilsson, Mats Gullberg, Ola Söderberg, Malin Jarvius ,Chatarina Larsson, and Jonas Jarvius

I. Need for Single-Molecule In Situ Analyses

787II. Problem of Specificity and Sensitivity

788III. Single- or Dual-Recognition Assays

789

IV. Some Possible Assay Formats

790V. Specific Detection of Macromolecules followed by Selectiv e

In Situ Amplification

794

VI. Future Directions

795References

795

35 . The Use of Subtelomeric Probes to Study Mental Retardatio n

Samantha J. L. Knight and Jonathan Flin t

I. Introduction

800II. Background

802III. Methods

806IV. Results

81 5

V. Critical Aspects of the Methodologies

81 5VI. Limitations, Pitfalls, and Misinterpretation of Data

820VII. Comparison with Other Methods

824VIII. Applications

825IX. Future Directions

826References

828

Index

83 3

Volumes in Series

869