Current Protocols in Cytometry
Table of Contents

1 Flow Cytometry Instrumentation

1.1 Overview of Flow Cytometry Instrumentation
1.2 Fluidics
1.3 Standardization, Calibration, and Control
1.4 Establishing and Maintaining System Linearity
1.5 Optical Filter Sets for Multiparameter Flow Cytometry
1.6 Laser Beam Shaping and Spot Size
1.7 High-Speed Cell Sorting
1.8 Principles of Gating
1.9 Lasers for Flow Cytometry
1.10 Techniques for Flow Cytometer Alignment
1.11 Flow Cytometers for Characterization of Microorganisms
1.12 Principles of Resonance Energy Transfer
1.13 Light Scatter: Detection and Usage
1.14 Compensation in Flow Cytometry
1.15 Time-Resolved Fluorescence Measurements
1.16 Simultaneous Analysis of the Cyan, Green, and Yellow Fluorescent Proteins
1.17 Plug Flow Cytometry
1.18 Dynamic Thermoregulation of the Sample in Flow Cytometry 
1.19 Excitation and Emission Spectra of Common Dyes

2 Image Cytometry Instrumentation

2.1 Contrast Enhancement in Light Microscopy
2.2 Microscope Objectives
2.3 Cameras
2.4 Optical Filtering Systems for Wavelength Selection in Light Microscopy
2.5 Digital Fluorescence Microscopy
2.6 Calibration: Sampling Density and Spatial Resolution
2.7 Microscope Alignment
2.8 Confocal Microscopy: Principles and Practices
2.9 Multiphoton Imaging
2.10 Scanning Laser Cytometry
2.11 Shading Correction: Compensation for Illumination and Sensor Inhomogeneities
2.12 Photobleaching Measurements of Diffusion in Cell Membranes and Aqueous Cell Compartments

3 Safety Procedures and Quality Control

3.1 Principles of Quality Control
3.2 Components of Quality Control
3.3 Testing the Efficiency of Aerosol Containment During Cell Sorting
3.4 Safe Use of Hazardous Chemicals 
3.5 Method for Visualizing Aerosol Contamination in Flow Sorters 

4 Molecular and Cellular Probes

4.1 Antibody Titration
4.2 Conjugation of Fluorochromes to Monoclonal Antibodies
4.3 Nucleic Acid Probes
4.4 Cellular Function Probes
4.5 Spectroscopic Analysis Using DNA and RNA Fluorescent Probes

5 Specimen Handling, Storage, and Preparation

5.1 Handling, Storage, and Preparation of Human Blood Cells
5.2 Handling, Storage, and Preparation of Human Tissues
5.3 Flow Analysis and Sorting of Plant Chromosomes

6 Phenotypic Analysis

6.1 Quality Control in Phenotypic Analysis by Flow Cytometry
6.2 Immunophenotyping
6.3 High-Sensitivity Immunoflourescence/Flow Cytomtery: Detection of Cytokine Receptors and Other Low-Abundance Membrane Molecules
6.4 Enumeration of CP34⁺ Hematopoietic Stem and Progenitor Cells (Revised, July 2003)
6.5 Immunophenotypic Analysis of Peripheral Blood Lymphocytes
6.6 Immunophenotypic Analysis of Human Mast Cells by Flow Cytometry
6.7 Measurement of CD40 Ligand (CD154) Expression on Resting and In Vitro-Activated T Cells
6.8 Enumeration of Absolute Cell Counts Using Immunophenotypic Techniques
6.9 Immunophenotypic Identification, Enumeration, and Characterization of Human Peripheral Blood Dendritic Cells and Dendritic-Cell Precursors
6.10 Immunophenotypic Analysis of Platelets 
6.11 Immunophenotypic Analysis of PNH Cells 
6.12 Quantitative Flow Cytometric Analysis of Membrane Antigen Expression 
6.13 Immunophenotyping Using a Laser Scanning Cytometer 
6.14 Enzymatic Amplification Staining for Cell Surface Antigens
6.15 Whole Blood Analysis of Leukocyte-Platelet Aggregates (New, April 2003)
6.16 Flow Cytometric Assessment of HLA Alloantibodies
6.17 Enumeration of Fetal Red Blood Cells, F Cells, and F Reticulocytes in Human Blood
6.18 Identification of Human Antigen-Specific T Cells Using MHC Class I and Class II Tetramers

7 Nucleic Acid Analysis

7.1 Overview of Nucleic Acid Analysis
7.2 Quality Control in Nucleic Acid Analysis
7.3 Differential Staining of DNA and RNA
7.4 Analysis of DNA Content and DNA Strand Breaks for Detection of Apoptotic Cells
7.5 DNA Content Measurement for DNA Ploidy and Cell Cycle Analysis
7.6 Analysis of Nuclear DNA Content and Ploidy in Higher Plants
7.7 Analysis of DNA Content and BrdU Incorporation
7.8 Analysis of DNA Denaturation
7.9 Bivariate Analysis of DNA Content and Expression of Cyclin Proteins
7.10 Flow Cytometric Analysis of Reticulated Platelets
7.11  Assessment of Viability, Immunofluorescence, and DNA Content
7.12 Flow Cytometric Analysis of RNA Synthesis by Detection of Bromouridine Incorporation
7.13 Sperm Chromatin Structure Assay for Fertility Assessment
7.14 Analysis of Cell Proliferation and Cell Survival by Continuous BrdU Labeling and Multivariate Flow Cytometry
7.15 Ultraviolet-Induced Detection of Halogenated Pyrimidines (UVID)
7.16 Analysis of DNA Content and Green Fluorescent Protein Expression
7.17 Analysis of Viral Infection and Viral and Cellular DNA and Proteins by Flow Cytometry 
7.18 Apoptosis Signaling Pathways 
7.19 Flow Cytometry of Apoptosis (Revised, July 2003)
7.20 Analysis of Fine-Needle Aspirate Biopsies from Solid Tumors by Laser Scanning Cytometry (LSC) 
7.21 Measurement of Cytogenetic Damage in Rodent Blood with a Single-Laser Flow Cytometer
7.22 Analysis of Tissue Imprints by Scanning Laser Cytometry
7.23 Cell Cycle Analysis of Budding Yeast Using SYTOX Green
7.24 Detection of Mitotic Cells
7.25 DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells
7.26 Assessment of Telomere Length, Phenotype, and DNA Content
7.27Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage (DNA Double-Strand Breaks)

8 Molecular Cytogenetics

8.1 Overview of Fluorescence In Situ Hybridization
8.2 Preparative Techniques
8.3 Probe Labeling and Fluorescence In Situ Hybridization
8.4 Immunocytochemical Detection
8.5 Processing and Staining of Cell and Tissue Material for Interphase Cytogenetics
8.6 Advanced Preparative Techniques to Establish Probes for Molecular Cytogenetics
8.7 Combination DNA/RNA FISH and Immunophenotyping
8.8 Single-Nucleotide Sequence Discrimination In Situ Using Padlock Probes
8.9 TSA Systems for the Enhancement of ISH Signals in Cytogenetics
8.10 Molecular Combing
8.11 Principles and Applications of PRINS in Cytogenetics 
8.12 Comparative Genomic Hybridization (CGH)-Detection of Unbalanced Genetic Aberrations Using Conventional and Micro-Array Techniques
8.13 Combined Immunofluorescence and FISH: New Prospects for Tumor Cell Detection/Identification

9 Studies of Cell Function

9.1 Overview of Functional Analysis
9.2 Assessment of Cell Viability
9.3 Measurements of Intracellular pH
9.4 Analysis of Intracellular Organelles by Flow Cytometry or Microscopy
9.5 Reporters of Gene Expression: Enzymatic Assays
9.6 Estimation of Membrane Potential by Flow Cytometry
9.7 Oxidative Metabolism
9.8 Measurement of Intracellular Calcium Ions by Flow Cytometry
9.9 Intracellular Cytokines
9.10 Assays of Natural Killer (NK) Cell Ligation to Target Cells
9.11 Flow Cytometric Analysis of Cell Division by Dye Dilution
9.12 Reporters of Gene Expression: Autofluorescent Proteins
9.13 In Vitro Invasion Assays: Phagocytosis of the Extracellular Matrix
9.14 Flow Cytometric Analysis of Mitochondrial Membrane Potential Using JC-1
9.15 Multiparameter Analysis of Physiological Changes in Apoptosis
9.16 Signal Transduction During Natural Killer Cell Activation
9.17 Assessment of Surface Markers and Functionality of Dendritic Cells (DCs) 
9.18 Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP)10
9.19 Assessment of Phagocyte Functions by Flow Cytometry 
9.20 Flow Cytometric Analysis of Calcium Mobilization in Whole-Blood Platelets (New, April 2003)
9.21 Flow Cytometric Analysis of Cytokine Responses in Stimulated Whole Blood: Simultaneous Quantitation of TNF-a-Secreting Cells and Soluble Cytokines (New, July 2003)

10 Data Processing and Analysis

10.1 Data Management
10.2 Data File Standard for Flow Cytometry, FCS 3.0
10.3 Listmode Data Processing
10.4 Multidimensional Data Analysis in Immunophenotyping
10.5 Two-Dimensional Image Processing and Analysis
10.6 Data Presentation
10.7 Data Analysis Through Modeling
10.8 Multivariate Analysis
10.9 Detection and Location of Hybridization Domains on Chromosomes by Image Cytometry
10.10 Three-Dimensional Image Visualization and Analysis
10.11 Image Processing and 2-D Morphometry
10.12 Dial-In Flow Cytometry Data Analysis 
10.13 The Application of Data Mining to Flow Cytometry 
10.14 Calibration and Shading Correction for Fluorescence Microscopes 
10.15 A Software Method for Color Compensation 

11 Microbiological Applications

11.1 Overview of Flow Cytometry and Microbiology
11.2 Flow Cytometry and Environmental Microbiology
11.3 Estimation of Microbial Viability Using Flow Cytometry
11.4 Sorting of Bacteria
11.5 Detection of Borreliacidal Antibodies by Flow Cytometry
11.6 Flow Cytometric Dectection of Pathogenic E. coli in Food
11.7 Mycobacterium tuberculosis Susceptibility Testing by Flow Cytometry
11.8 Antibiotic Susceptibility Testing by Flow Cytometry
11.9 Determination of Bacterial Biomass from Flow Cytometric Measurements of Forward Light Scatter Intensity
11.10 Flow Cytometry of Yeasts
11.11 Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples
11.12 DNA/RNA Analysis of Phytoplankton by Flow Cytometry
11.13 Cell Cycle Analysis of Yeasts
11.14 Flow Cytometric Assessment of Drug Susceptibility in Leishmania infantum Promastigotes
11.15 Resolution of Viable and Membrane-Compromised Free Bacteria in Aquatic Environments by Flow Cytometry
11.16 Functional Assays of Oxidative Stress Using Genetically Engineered Escherichia coli Strains (New, April 2003)
11.17 Labeling of Bacterial Pathogens for Flow Cytometric Detection and Enumeration

Appendix 1 Abbreviations and Useful Data
1A Abbreviations Used in this Manual
1B Common Conversion Factors

Appendix 2 Stock Solutions, Equipment, and Laboratory Guidelines
2A Common Stock Solutions, Buffers, and Media

Appendix 3 Commonly Used Techniques
3A Cell Counting HTML PDF (Size 79KB)
3B Techniques for Mammalian Cell Tissue Culture
3C Diagnosis and Treatment of Mycoplasma-Contaminated Cell Cultures
3D Wright-Giemsa and Nonspecific Esterase Staining of Cells
3E Techniques for Bacterial Cell Culture: Media Preparation and Bacteriological Tools
3F Growing Bacteria in Liquid Media
3G Growing Bacteria on Solid Media
3H Importing Biological Materials

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