Current Protocols in Cytometry

INTRODUCTION......Page 14
1.1 Overview of Flow Cytometry Instrumentation......Page 17
1.2 Fluidics......Page 25
1.3 Standardization, Calibration, and Control in Flow Cytometry......Page 32
1.4 Establishing and Maintaining System Linearity......Page 53
1.5 Optical Filter Sets for Multiparameter Flow Cytometry......Page 65
1.6 Laser Beam Shaping and Spot Size......Page 73
1.7 High-Speed Cell Sorting......Page 78
1.8 Principles of Gating......Page 85
1.9 Lasers for Flow Cytometry......Page 97
1.10 Techniques for Flow Cytometer Alignment......Page 113
1.11 Flow Cytometers for Characterization of Microorganisms......Page 119
1.12 Principles of Resonance Energy Transfer......Page 128
1.13 Light Scatter: Detection and Usage......Page 141
1.14 Compensation in Flow Cytometry......Page 149
1.15 Time-Resolved Fluorescence Measurements......Page 169
1.16 Simultaneous Analysis of the Cyan, Green, and Yellow Fluorescent Proteins......Page 185
1.17 Plug Flow Cytometry......Page 197
1.18 Dynamic Thermoregulation of the Sample in Flow Cytometry......Page 207
1.19 Excitation and Emission Spectra of Common Dyes......Page 223
INTRODUCTION......Page 230
2.1 Contrast Enhancement in Light Microscopy......Page 232
2.2 Microscope Objectives......Page 243
2.3 Cameras......Page 257
2.4 Optical Filtering Systems for Wavelength Selection in Light Microscopy......Page 272
2.5 Digital Fluorescence Microscopy......Page 283
2.6 Calibration: Sampling Density and Spatial Resolution......Page 288
2.7 Microscope Alignment......Page 303
2.8 Confocal Microscopy: Principles and Practices......Page 311
2.9 Multiphoton Imaging......Page 323
2.10 Scanning Laser Cytometry......Page 334
2.11 Shading Correction: Compensation for Illumination and Sensor Inhomogeneities......Page 343
2.12 Photobleaching Measurements of Diffusion in Cell Membranes and Aqueous Cell Compartments......Page 355
INTRODUCTION......Page 370
3.1 Principles of Quality Control......Page 372
3.2 Components of Quality Control......Page 374
3.3 Testing the Efficiency of Aerosol Containment During Cell Sorting......Page 378
3.4 Safe Use of Hazardous Chemicals......Page 393
3.5 Method for Visualizing Aerosol Contamination in Flow Sorters......Page 426
INTRODUCTION......Page 433
4.1 Titering Antibodies......Page 435
4.2 Conjugation of Fluorochromes to Monoclonal Antibodies......Page 448
4.3 Nucleic Acid Probes......Page 460
4.4 Cellular Function Probes......Page 470
4.5 Spectroscopic Analysis Using DNA and RNA Fluorescent Probes......Page 487
INTRODUCTION......Page 495
5.1 Handling, Storage, and Preparation of Human Blood Cells......Page 496
5.3 Flow Analysis and Sorting of Plant Chromosomes......Page 509
INTRODUCTION......Page 557
6.1 Quality Control in Phenotypic Analysis by Flow Cytometry......Page 559
6.2 Immunophenotyping......Page 563
6.3 High-Sensitivity Immunofluorescence/Flow Cytometry: Detection of Cytokine Receptors and Other Low-Abundance Membrane Molecules......Page 581
6.4 Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells......Page 594
6.5 Immunophenotypic Analysis of Peripheral Blood Lymphocytes......Page 617
6.6 Immunophenotypic Analysis of Human Mast Cells by Flow Cytometry......Page 631
6.7 Measurement of CD40 Ligand (CD154) Expression on Resting and In Vitro-Activated T Cells......Page 649
6.8 Enumeration of Absolute Cell Counts Using Immunophenotypic Techniques......Page 659
6.9 Immunophenotypic Identification, Enumeration, and Characterization of Human Peripheral Blood Dendritic Cells and Dendritic-Cell Precursors......Page 685
6.10 Immunophenotypic Analysis of Platelets......Page 698
6.11 Immunophenotypic Analysis of PNH Cells......Page 715
6.12 Quantitative Flow Cytometric Analysis of Membrane Antigen Expression......Page 731
6.13 Immunophenotyping Using a Laser Scanning Cytometer......Page 753
6.14 Enzymatic Amplification Staining for Cell Surface Antigens......Page 768
6.15 Whole Blood Analysis of Leukocyte-Platelet Aggregates......Page 778
6.16 Flow Cytometric Assessment of HLA Alloantibodies......Page 786
6.17 Enumeration of Fetal Red Blood Cells, F Cells, and F Reticulocytes in Human Blood......Page 810
6.18 Identification of Human Antigen-Specific T Cells Using MHC Class I and Class II Tetramers......Page 826
6.19 ZAP-70 Staining in Chronic Lymphocytic Leukemia......Page 838
6.20 Multiparameter Analysis of Intracellular Phosphoepitopes in Immunophenotyped Cell Populations by Flow Cytometry......Page 852
INTRODUCTION......Page 874
7.1 Overview of Nucleic Acid Analysis......Page 880
7.2 Quality Control in Nucleic Acid Analysis......Page 884
7.3 Differential Staining of DNA and RNA......Page 889
7.4 Analysis of DNA Content and DNA Strand Breaks for Detection of Apoptotic Cells......Page 905
7.5 DNA Content Measurement for DNA Ploidy and Cell Cycle Analysis......Page 913
7.6 Analysis of Nuclear DNA Content and Ploidy in Higher Plants......Page 937
7.7 Analysis of DNA Content and BrdU Incorporation......Page 959
7.8 Analysis of DNA Denaturation......Page 968
7.9 Bivariate Analysis of DNA Content and Expression of Cyclin Proteins......Page 977
7.10 Flow Cytometric Analysis of Reticulated Platelets......Page 983
7.11 Assessment of Viability, Immunofluorescence, and DNA Content......Page 989
7.12 Flow Cytometric Analysis of RNA Synthesis by Detection of Bromouridine Incorporation......Page 998
7.13 Sperm Chromatin Structure Assay for Fertility Assessment......Page 1009
7.14 Analysis of Cell Proliferation and Cell Survival by Continuous BrdU Labeling and Multivariate Flow Cytometry......Page 1036
7.15 Ultraviolet-Induced Detection of Halogenated Pyrimidines (UVID)......Page 1045
7.16 Analysis of DNA Content and Green Fluorescent Protein Expression......Page 1051
7.17 Analysis of Viral Infection and Viral and Cellular DNA and Proteins by Flow Cytometry......Page 1061
7.18 Apoptosis Signaling Pathways......Page 1070
7.19 Flow Cytometry of Apoptosis......Page 1080
7.20 Analysis of Fine-Needle Aspirate Biopsies from Solid Tumors by Laser Scanning Cytometry (LSC)......Page 1113
7.21 Measurement of Cytogenetic Damage in Rodent Blood with a Single-Laser Flow Cytometer......Page 1123
7.22 Analysis of Tissue Imprints by Scanning Laser Cytometry......Page 1132
7.23 Cell Cycle Analysis of Budding Yeast Using SYTOX Green......Page 1138
7.24 Detection of Mitotic Cells......Page 1142
7.25 DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells......Page 1149
7.26 Assessment of Telomere Length, Phenotype, and DNA Content......Page 1160
7.27 Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage (DNA Double-Strand Breaks)......Page 1173
INTRODUCTION......Page 1180
8.1 Overview of Fluorescence In Situ Hybridization Techniques for Molecular Cytogenetics......Page 1183
8.2 Basic Preparative Techniques for Fluorescence In Situ Hybridization......Page 1189
8.3 Probe Labeling and Fluorescence In Situ Hybridization......Page 1212
8.4 Immunocytochemical Detection......Page 1233
8.5 Processing and Staining of Cell and Tissue Material for Interphase Cytogenetics......Page 1251
8.6 Advanced Preparative Techniques to Establish Probes for Molecular Cytogenetics......Page 1273
8.7 Combination DNA/RNA Fish and Immunophenotyping......Page 1296
8.8 Single-Nucleotide Sequence Discrimination In Situ Using Padlock Probes......Page 1310
8.9 Tyramide Signal Amplification (TSA) Systems for the Enhancement of ISH Signals in Cytogenetics......Page 1322
8.10 Molecular Combing......Page 1338
8.11 Principles and Applications of PRINS in Cytogenetics......Page 1361
8.12 Comparative Genomic Hybridization (CGH)-Detection of Unbalanced Genetic Aberrations Using Conventional and Micro-Array Techniques......Page 1374
8.13 Combined Immunofluorescence and FISH: New Prospects for Tumor Cell Detection/Identification......Page 1404
INTRODUCTION......Page 1415
9.1 Overview of Functional Cell Assays......Page 1417
9.2 Assessment of Cell Viability......Page 1423
9.3 Flow Cytometric Measurement of Intracellular pH......Page 1437
9.4 Analysis of Intracellular Organelles by Flow Cytometry or Microscopy......Page 1447
9.5 Reporters of Gene Expression: Enzymatic Assays......Page 1471
9.6 Estimation of Membrane Potential by Flow Cytometry......Page 1493
9.7 Oxidative Metabolism......Page 1505
9.8 Measurement of Intracellular Calcium Ions by Flow Cytometry......Page 1519
9.9 Intracellular Cytokines......Page 1538
9.10 Assays of Natural Killer (NK) Cell Ligation to Target Cells......Page 1549
9.11 Flow Cytometric Analysis of Cell Division by Dye Dilution......Page 1557
9.12 Reporters of Gene Expression: Autofluorescent Proteins......Page 1567
9.13 In Vitro Invasion Assays: Phagocytosis of the Extracellular Matrix......Page 1583
9.14 Flow Cytometric Analysis of Mitochondrial Membrane Potential Using JC-1......Page 1591
9.15 Multiparameter Analysis of Physiological Changes in Apoptosis......Page 1598
9.16 Signal Transduction During Natural Killer Cell Activation......Page 1605
9.17 Assessment of Surface Markers and Functionality of Dendritic Cells (DCs)......Page 1618
9.18 Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP)......Page 1633
9.19 Assessment of Phagocyte Functions by Flow Cytometry......Page 1644
9.20 Flow Cytometric Analysis of Calcium Mobilization in Whole-Blood Platelets......Page 1666
9.21 Flow Cytometric Analysis of Cytokine Responses in Stimulated Whole Blood: Simultaneous Quantitation of TNF-α-Secreting Cells and Soluble Cytokines......Page 1674
INTRODUCTION......Page 1695
10.1 Data Management......Page 1697
10.2 Data File Standard for Flow Cytometry, FCS 3.0......Page 1703
10.3 Listmode Data Processing......Page 1708
10.4 Multidimensional Data Analysis in Immunophenotyping......Page 1713
10.5 Two-Dimensional Image Processing and Analysis......Page 1720
10.6 Data Presentation......Page 1734
10.7 Data Analysis Through Modeling......Page 1741
10.8 Multivariate Analysis......Page 1761
10.9 Detection and Location of Hybridization Domains on Chromosomes by Image Cytometry......Page 1782
10.10 Three-Dimensional Image Visualization and Analysis......Page 1793
10.11 Image Processing and 2-D Morphometry......Page 1806
10.12 Dial-In Flow Cytometry Data Analysis......Page 1817
10.13 The Application of Data Mining to Flow Cytometry......Page 1824
10.14 Calibration and Shading Correction for Fluorescence Microscopes......Page 1830
10.15 A Software Method for Color Compensation......Page 1837
INTRODUCTION......Page 1849
11.1 Overview of Flow Cytometry and Microbiology......Page 1852
11.2 Flow Cytometry and Environmental Microbiology......Page 1856
11.3 Estimation of Microbial Viability Using Flow Cytometry......Page 1869
11.4 Sorting of Bacteria......Page 1890
11.5 Detection of Borreliacidal Antibodies by Flow Cytometry......Page 1902
11.6 Flow Cytometric Detection of Pathogenic E. coli in Food......Page 1914
11.7 Mycobacterium tuberculosis Susceptibility Testing by Flow Cytometry......Page 1923
11.8 Antibiotic Susceptibility Testing by Flow Cytometry......Page 1931
11.9 Determination of Bacterial Biomass from Flow Cytometric Measurements of Forward Light Scatter Intensity......Page 1940
11.10 Flow Cytometry of Yeasts......Page 1950
11.11 Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples......Page 1958
11.12 DNA/RNA Analysis of Phytoplankton by Flow Cytometry......Page 1973
11.13 Cell Cycle Analysis of Yeasts......Page 1987
11.14 Flow Cytometric Assessment of Drug Susceptibility in Leishmania infantum Promastigotes......Page 1996
11.15 Resolution of Viable and Membrane-Compromised Free Bacteria in Aquatic Environments by Flow Cytometry......Page 2005
11.16 Functional Assays of Oxidative Stress Using Genetically Engineered Escherichia coli Strains......Page 2012
11.17 Labeling of Bacterial Pathogens for Flow Cytometric Detection and Enumeration......Page 2021
INTRODUCTION......Page 2041
12.1 Comparative Overview of Flow and Image Cytometry......Page 2043
12.2 Basics of Digital Microscopy......Page 2054
12.3 Modern Confocal Microscopy......Page 2068
12.4 Time-Lapse Microscopy Approaches to Track Cell Cycle Progression at the Single-Cell Level......Page 2080
12.5 Three-Dimensional Visualization of Blood and Lymphatic Vasculature in Tissue Whole Mounts Using Confocal Microscopy......Page 2091
1A Abbreviations Used in This Manual......Page 2102
1B Common Conversion Factors......Page 2107
2A Common Stock Solutions, Buffers, and Media......Page 2115
3A Cell Counting......Page 2119
3B Techniques for Mammalian Cell Tissue Culture......Page 2123
3C Diagnosis and Treatment of Mycoplasma-Contaminated Cell Cultures......Page 2133
3D Wright-Giemsa and Nonspecific Esterase Staining of Cells......Page 2137
3E Techniques for Bacterial Cell Culture: Media Preparation and Bacteriological Tools......Page 2141
3F Growing Bacteria in Liquid Media......Page 2149
3G Growing Bacteria on Solid Media......Page 2151
3H Importing Biological Materials......Page 2156
SELECTED SUPPLIERS OF REAGENTS AND EQUIPMENT......Page 2159