Current Protocols in Cytometry

Current Protocols in Cytometry

Online ISBN: 9780471142959

DOI: 10.1002/0471142956

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  1. Preface
  2. Foreword
  3. Selected Suppliers of Reagents and Equipment
  4. Chapter 1 Flow Cytometry Instrumentation
    1. Introduction
    2. UNIT 1.1 Overview of Flow Cytometry Instrumentation
    3. UNIT 1.2 Fluidics
    4. UNIT 1.3 Standardization, Calibration, and Control in Flow Cytometry
    5. UNIT 1.4 Establishing and Maintaining System Linearity
    6. UNIT 1.5 Optical Filter Sets for Multiparameter Flow Cytometry
    7. UNIT 1.6 Laser Beam Shaping and Spot Size
    8. UNIT 1.7 High-Speed Cell Sorting
    9. UNIT 1.8 Principles of Gating
    10. UNIT 1.9 Lasers for Flow Cytometry
    11. UNIT 1.10 Techniques for Flow Cytometer Alignment
    12. UNIT 1.11 Flow Cytometers for Characterization of Microorganisms
    13. UNIT 1.12 Principles of Resonance Energy Transfer
    14. UNIT 1.13 Light Scatter: Detection and Usage
    15. UNIT 1.14 Compensation in Flow Cytometry
    16. UNIT 1.15 Time-Resolved Fluorescence Measurements
    17. UNIT 1.16 Simultaneous Analysis of the Cyan, Green, and Yellow Fluorescent Proteins
    18. UNIT 1.17 Plug Flow Cytometry
    19. UNIT 1.18 Dynamic Thermoregulation of the Sample in Flow Cytometry
    20. UNIT 1.19 Excitation and Emission Spectra of Common Dyes
    21. UNIT 1.20 Characterization of Flow Cytometer Instrument Sensitivity
    22. UNIT 1.21 Separation Index: An Easy-to-Use Metric for Evaluation of Different Configurations on the Same Flow Cytometer
    23. UNIT 1.22 Fundamentals of Acoustic Cytometry
    24. UNIT 1.23 Pulse Width for Particle Sizing
    25. UNIT 1.24 Practical Issues in High-Speed Cell Sorting
    26. UNIT 1.25 Capture of Fluorescence Decay Times by Flow Cytometry
    27. UNIT 1.26 Fountain Flow Cytometry
    28. UNIT 1.27 Spectral Flow Cytometry
    29. UNIT 1.28 Evaluation and Purchase of an Analytical Flow Cytometer: Some of the Numerous Factors to Consider
  5. Chapter 2 Image Cytometry Instrumentation
    1. Introduction
    2. UNIT 2.1 Contrast Enhancement in Light Microscopy
    3. UNIT 2.2 Microscope Objectives
    4. UNIT 2.3 Light Microscopy Digital Imaging
    5. UNIT 2.4 Optical Filters for Wavelength Selection in Fluorescence Instrumentation
    6. UNIT 2.5 Digital Fluorescence Microscopy
    7. UNIT 2.6 Calibration: Sampling Density and Spatial Resolution
    8. UNIT 2.7 Microscope Alignment
    9. UNIT 2.8 Confocal Microscopy: Principles and Practices
    10. UNIT 2.9 Multi-Photon Imaging
    11. UNIT 2.10 Scanning Laser Cytometry
    12. UNIT 2.11 Shading Correction: Compensation for Illumination and Sensor Inhomogeneities
    13. UNIT 2.12 Photobleaching Measurements of Diffusion in Cell Membranes and Aqueous Cell Compartments
    14. UNIT 2.13 Optimizing Laser Source Operation for Confocal and Multiphoton Laser Scanning Microscopy
    15. UNIT 2.14 Methods and Applications of Laser-Enabled Analysis and Processing (LEAP)
    16. UNIT 2.15 Measurement of Molecular Mobility with Fluorescence Correlation Spectroscopy
    17. UNIT 2.16 Evaluation and Purchase of Confocal Microscopes: Numerous Factors to Consider
    18. UNIT 2.17 Super-Resolution Microscopy: A Comparative Treatment
    19. UNIT 2.18 Quantitative Fluorescent Speckle Microscopy (QFSM) to Measure Actin Dynamics
    20. UNIT 2.19 Analysis of Protein and Lipid Dynamics Using Confocal Fluorescence Recovery After Photobleaching (FRAP)
    21. UNIT 2.20 Comparative and Practical Aspects of Localization-Based Super-Resolution Imaging
    22. UNIT 2.21 Building a Live Cell Microscope: What You Need and How to Do It
    23. UNIT 2.22 How to Build a Time-Gated Luminescence Microscope
  6. Chapter 3 Safety Procedures and Quality Control
    1. Introduction
    2. UNIT 3.1 Principles of Quality Control
    3. UNIT 3.2 Components of Quality Control
    4. UNIT 3.3 Testing the Efficiency of Aerosol Containment During Cell Sorting
    5. UNIT 3.4 Safe Use of Hazardous Chemicals
    6. UNIT 3.5 Method for Visualizing Aerosol Contamination in Flow Sorters
    7. UNIT 3.6 Standard Safety Practices for Sorting of Unfixed Cells
  7. Chapter 4 Molecular and Cellular Probes
    1. Introduction
    2. UNIT 4.1 Titering Antibodies
    3. UNIT 4.2 Conjugation of Fluorochromes to Monoclonal Antibodies
    4. UNIT 4.3 Nucleic Acid Probes
    5. UNIT 4.4 Cellular Function Probes
    6. UNIT 4.5 Spectroscopic Analysis Using DNA and RNA Fluorescent Probes
    7. UNIT 4.6 Flow Cytometric Sorting of Bacterial Surface-Displayed Libraries
    8. UNIT 4.7 Construction and Screening of Antigen Targeted Immune Yeast Surface Display Antibody Libraries
  8. Chapter 5 Specimen Handling, Storage, and Preparation
    1. Introduction
    2. UNIT 5.1 Handling, Storage, and Preparation of Human Blood Cells
    3. UNIT 5.2 Handling, Storage, and Preparation of Human Tissues
    4. UNIT 5.3 Flow Analysis and Sorting of Plant Chromosomes
  9. Chapter 6 Phenotypic Analysis
    1. Introduction
    2. UNIT 6.1 Quality Control in Phenotypic Analysis by Flow Cytometry
    3. UNIT 6.2 Immunophenotyping
    4. UNIT 6.3 High-Sensitivity Immunofluorescence/Flow Cytometry: Detection of Cytokine Receptors and Other Low-Abundance Membrane Molecules
    5. UNIT 6.4 Enumeration of CD34+ Hematopoietic Stem and Progenitor Cells
    6. UNIT 6.5 Immunophenotypic Analysis of Peripheral Blood Lymphocytes
    7. UNIT 6.6 Immunophenotypic Analysis of Human Mast Cells by Flow Cytometry
    8. UNIT 6.7 Measurement of CD40 Ligand (CD154) Expression on Resting and In Vitro–Activated T Cells
    9. UNIT 6.8 Enumeration of Absolute Cell Counts Using Immunophenotypic Techniques
    10. UNIT 6.9 Immunophenotypic Identification, Enumeration, and Characterization of Human Peripheral Blood Dendritic Cells and Dendritic-Cell Precursors
    11. UNIT 6.10 Immunophenotypic Analysis of Platelets
    12. UNIT 6.11 Immunophenotypic Analysis of PNH Cells
    13. UNIT 6.12 Quantitative Flow Cytometric Analysis of Membrane Antigen Expression
    14. UNIT 6.13 Immunophenotyping Using a Laser Scanning Cytometer
    15. UNIT 6.14 Enzymatic Amplification Staining for Cell Surface Antigens
    16. UNIT 6.15 Whole Blood Analysis of Leukocyte-Platelet Aggregates
    17. UNIT 6.16 Flow Cytometric Assessment of HLA Alloantibodies
    18. UNIT 6.17 Enumeration of Fetal Red Blood Cells, F Cells, and F Reticulocytes in Human Blood
    19. UNIT 6.18 Identification of Human Antigen-Specific T Cells Using MHC Class I and Class II Tetramers
    20. UNIT 6.19 ZAP-70 Staining in Chronic Lymphocytic Leukemia
    21. UNIT 6.20 Multiparameter Analysis of Intracellular Phosphoepitopes in Immunophenotyped Cell Populations by Flow Cytometry
    22. UNIT 6.21 Ten-Color Immunophenotyping of Hematopoietic Cells
    23. UNIT 6.22 Flow Cytometric Screening for the HLA-B27 Antigen on Peripheral Blood Lymphocytes
    24. UNIT 6.23 Immunophenotyping of Plasma Cells
    25. UNIT 6.24 Flow Rate Calibration for Absolute Cell Counting Rationale and Design
    26. UNIT 6.25 Flow Cytometric Immunophenotyping of Cerebrospinal Fluid
    27. UNIT 6.26 Calibration of Flow Cytometry for Quantitative Quantum Dot Measurements
    28. UNIT 6.27 Assessment of Beta Cell Viability
    29. UNIT 6.28 Measurement of T Cell Activation After 16-hr In Vitro Stimulation with Concanavalin A
    30. UNIT 6.29 Titration of Fluorochrome-Conjugated Antibodies for Labeling Cell Surface Markers on Live Cells
    31. UNIT 6.30 Phenotypic Analysis Using Very Small Volumes of Blood
    32. UNIT 6.31 Fluorescent Cell Barcoding for Multiplex Flow Cytometry
    33. UNIT 6.32 Quantitative Assessment of Pancreatic Islets Using Laser Scanning Cytometry
    34. UNIT 6.33 Three-Dimensional Second-Harmonic Generation Imaging of Fibrillar Collagen in Biological Tissues
    35. UNIT 6.34 Flow Cytometry–Based Cytotoxicity and Antibody Binding Assay
  10. Chapter 7 Nucleic Acid Analysis
    1. Introduction
    2. UNIT 7.1 Overview of Nucleic Acid Analysis
    3. UNIT 7.2 Critical Aspects in Analysis of Cellular DNA Content
    4. UNIT 7.3 Differential Staining of DNA and RNA
    5. UNIT 7.4 Analysis of DNA Content and DNA Strand Breaks for Detection of Apoptotic Cells
    6. UNIT 7.5 DNA Content Measurement for DNA Ploidy and Cell Cycle Analysis
    7. UNIT 7.6 Analysis of Nuclear DNA Content and Ploidy in Higher Plants
    8. UNIT 7.7 Analysis of DNA Content and BrdU Incorporation
    9. UNIT 7.8 Analysis of DNA Denaturation
    10. UNIT 7.9 Bivariate Analysis of DNA Content and Expression of Cyclin Proteins
    11. UNIT 7.10 Flow Cytometric Analysis of Reticulated Platelets
    12. UNIT 7.11 Assessment of Viability, Immunofluorescence, and DNA Content
    13. UNIT 7.12 Flow Cytometric Analysis of RNA Synthesis by Detection of Bromouridine Incorporation
    14. UNIT 7.13 Sperm Chromatin Structure Assay for Fertility Assessment
    15. UNIT 7.14 Analysis of Cell Proliferation and Cell Survival by Continuous BrdU Labeling and Multivariate Flow Cytometry
    16. UNIT 7.15 Ultraviolet-Induced Detection of Halogenated Pyrimidines (UVID)
    17. UNIT 7.16 Analysis of DNA Content and Green Fluorescent Protein Expression
    18. UNIT 7.17 Analysis of Viral Infection and Viral and Cellular DNA and Proteins by Flow Cytometry
    19. UNIT 7.18 Apoptosis Signaling Pathways
    20. UNIT 7.19 Flow Cytometry of Apoptosis
    21. UNIT 7.20 Analysis of Fine-Needle Aspirate Biopsies from Solid Tumors by Laser Scanning Cytometry (LSC)
    22. UNIT 7.21 Measurement of Cytogenetic Damage in Rodent Blood with a Single-Laser Flow Cytometer
    23. UNIT 7.22 Analysis of Tissue Imprints by Scanning Laser Cytometry
    24. UNIT 7.23 Cell Cycle Analysis of Budding Yeast Using SYTOX Green
    25. UNIT 7.24 Detection of Mitotic Cells
    26. UNIT 7.25 DRAQ5 Labeling of Nuclear DNA in Live and Fixed Cells
    27. UNIT 7.26 Assessment of Telomere Length, Phenotype, and DNA Content
    28. UNIT 7.27 Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage (DNA Double-Strand Breaks)
    29. UNIT 7.28 RNA and DNA Aptamers in Cytomics Analysis
    30. UNIT 7.29 Nuclear DNA Content Analysis of Plant Seeds by Flow Cytometry
    31. UNIT 7.30 Estimation of Relative Nuclear DNA Content in Dehydrated Plant Tissues by Flow Cytometry
    32. UNIT 7.31 Assessment of Cell Proliferation by 5-Bromodeoxyuridine (BrdU) Labeling for Multicolor Flow Cytometry
    33. UNIT 7.32 Polychromatic Analysis of Mitochondrial Membrane Potential Using JC-1
    34. UNIT 7.33 SYTO Probes: Markers of Apoptotic Cell Demise
    35. UNIT 7.34 Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining
    36. UNIT 7.35 Assessment of Histone Acetylation Levels in Relation to Cell Cycle Phase
    37. UNIT 7.36 Click-iT Proliferation Assay with Improved DNA Histograms
    38. UNIT 7.37 High-Resolution Multiparameter DNA Flow Cytometry for the Detection and Sorting of Tumor and Stromal Subpopulations from Paraffin-Embedded Tissues
    39. UNIT 7.38 Dual-Pulse Labeling Using 5-Ethynyl-2′-Deoxyuridine (EdU) and 5-Bromo-2′-Deoxyuridine (BrdU) in Flow Cytometry
    40. UNIT 7.39 High-Resolution Cell Cycle and DNA Ploidy Analysis in Tissue Samples
    41. UNIT 7.40 Zinc Fixation for Flow Cytometry Analysis of Intracellular and Surface Epitopes, DNA Content, and Cell Proliferation
  11. Chapter 8 Molecular Cytogenetics
    1. Introduction
    2. UNIT 8.1 Overview of Fluorescence In Situ Hybridization Techniques for Molecular Cytogenetics
    3. UNIT 8.2 Basic Preparative Techniques for Fluorescence In Situ Hybridization
    4. UNIT 8.3 Probe Labeling and Fluorescence In Situ Hybridization
    5. UNIT 8.4 Immunocytochemical Detection
    6. UNIT 8.5 Processing and Staining of Cell and Tissue Material for Interphase Cytogenetics
    7. UNIT 8.6 Advanced Preparative Techniques to Establish Probes for Molecular Cytogenetics
    8. UNIT 8.7 Combination DNA/RNA Fish and Immunophenotyping
    9. UNIT 8.8 Single-Nucleotide Sequence Discrimination In Situ Using Padlock Probes
    10. UNIT 8.9 Tyramide Signal Amplification (TSA) Systems for the Enhancement of ISH Signals in Cytogenetics
    11. UNIT 8.10 Molecular Combing
    12. UNIT 8.11 Principles and Applications of PRINS in Cytogenetics
    13. UNIT 8.12 Comparative Genomic Hybridization (CGH)—Detection of Unbalanced Genetic Aberrations Using Conventional and Micro-Array Techniques
    14. UNIT 8.13 Combined Immunofluorescence and FISH: New Prospects for Tumor Cell Detection/Identification
    15. UNIT 8.14 Application of Flow-FISH for Dynamic Measurement of Telomere Length in Cell Division
  12. Chapter 9 Studies of Cell Function
    1. Introduction
    2. UNIT 9.1 Overview of Functional Cell Assays
    3. UNIT 9.2 Assessment of Cell Viability
    4. UNIT 9.3 Flow Cytometric Measurement of Intracellular pH
    5. UNIT 9.4 Analysis of Intracellular Organelles by Flow Cytometry or Microscopy
    6. UNIT 9.5 Reporters of Gene Expression: Enzymatic Assays
    7. UNIT 9.6 Estimation of Membrane Potential by Flow Cytometry
    8. UNIT 9.7 Oxidative Metabolism
    9. UNIT 9.8 Measurement of Intracellular Calcium Ions by Flow Cytometry
    10. UNIT 9.9 Intracellular Cytokines
    11. UNIT 9.10 Assays of Natural Killer (NK) Cell Ligation to Target Cells
    12. UNIT 9.11 Flow Cytometric Analysis of Cell Division by Dilution of CFSE and Related Dyes
    13. UNIT 9.12 Reporters of Gene Expression: Autofluorescent Proteins
    14. UNIT 9.13 In Vitro Invasion Assays: Phagocytosis of the Extracellular Matrix
    15. UNIT 9.14 Flow Cytometric Analysis of Mitochondrial Membrane Potential Using JC-1
    16. UNIT 9.15 Multiparameter Analysis of Physiological Changes in Apoptosis
    17. UNIT 9.16 Signal Transduction During Natural Killer Cell Activation
    18. UNIT 9.17 Assessment of Surface Markers and Functionality of Dendritic Cells (DCs)
    19. UNIT 9.18 Stem Cell Identification and Sorting Using the Hoechst 33342 Side Population (SP)
    20. UNIT 9.19 Assessment of Phagocyte Functions by Flow Cytometry
    21. UNIT 9.20 Flow Cytometric Analysis of Calcium Mobilization in Whole-Blood Platelets
    22. UNIT 9.21 Flow Cytometric Analysis of Cytokine Responses in Stimulated Whole Blood: Simultaneous Quantitation of TNF-α-Secreting Cells and Soluble Cytokines
    23. UNIT 9.22 Optimized Whole-Blood Assay for Measurement of ZAP-70 Protein Expression
    24. UNIT 9.23 Flow Cytometry of the Side Population (SP)
    25. UNIT 9.24 High-Throughput Cytotoxicity Screening by Propidium Iodide Staining
    26. UNIT 9.25 Advanced Application of CFSE for Cellular Tracking
    27. UNIT 9.26 Immunophenotyping and DNA Content Analysis of Acetone-Fixed Cells
    28. UNIT 9.27 Whole Blood Processing for Measurement of Signaling Proteins by Flow Cytometry
    29. UNIT 9.28 Measurement of Cytoplasmic to Nuclear Translocation
    30. UNIT 9.29 Overview of Very Small Embryonic-Like Stem Cells (VSELs) and Methodology of Their Identification and Isolation by Flow Cytometric Methods
    31. UNIT 9.30 Stem Cell Side Population Analysis and Sorting Using DyeCycle Violet
    32. UNIT 9.31 Measurement of Phagocytosis and of the Phagosomal Environment in Polymorphonuclear Phagocytes by Flow Cytometry
    33. UNIT 9.32 Yeast Cell Cycle Analysis: Combining DNA Staining with Cell and Nuclear Morphology
    34. UNIT 9.33 Identification of Endothelial Cells and Progenitor Cell Subsets in Human Peripheral Blood
    35. UNIT 9.34 Amine-Reactive Dyes for Dead Cell Discrimination in Fixed Samples
    36. UNIT 9.35 Detection of Intracellular Glutathione Using ThiolTracker Violet Stain and Fluorescence Microscopy
    37. UNIT 9.36 In Situ Proximity Ligation Assay for Microscopy and Flow Cytometry
    38. UNIT 9.37 Assessing Mitochondrial Redox Status by Flow Cytometric Methods: Vascular Response to Fluid Shear Stress
    39. UNIT 9.38 A Violet Ratiometric Membrane Probe for the Detection of Apoptosis
    40. UNIT 9.39 Ex Vivo Imaging of Excised Tissue Using Vital Dyes and Confocal Microscopy
    41. UNIT 9.40 Flow Cytometry-Based Quantification of Cell Proliferation in the Mixed Cell Co-Culture
    42. UNIT 9.41 Kinetic Viability Assays Using DRAQ7 Probe
    43. UNIT 9.42 Multiparameter Analysis of Apoptosis Using Lab-on-a-Chip Flow Cytometry
    44. UNIT 9.43 Real-Time Detection of Protein Trafficking with High-Throughput Flow Cytometry (HTFC) and Fluorogen-Activating Protein (FAP) Base Biosensor
    45. UNIT 9.44 OpenSource Lab-on-a-Chip Physiometer for Accelerated Zebrafish Embryo Biotests
    46. Unit 9.45 Measurement of Autophagy by Flow Cytometry
    47. Unit 9.46 Immunophenotyping of Paucicellular Samples
    48. UNIT 9.47 Attenuation of Replication Stress–Induced Premature Cellular Senescence to Assess Anti-Aging Modalities
  13. Chapter 10 Data Processing and Analysis
    1. Introduction
    2. UNIT 10.1 Data Management
    3. UNIT 10.2 Data File Standard for Flow Cytometry, FCS 3.0
    4. UNIT 10.3 Listmode Data Processing
    5. UNIT 10.4 Multidimensional Data Analysis in Immunophenotyping
    6. UNIT 10.5 Two-Dimensional Image Processing and Analysis
    7. UNIT 10.6 Data Presentation
    8. UNIT 10.7 Data Analysis Through Modeling
    9. UNIT 10.8 Multivariate Analysis
    10. UNIT 10.9 Detection and Location of Hybridization Domains on Chromosomes by Image Cytometry
    11. UNIT 10.10 Three-Dimensional Image Visualization and Analysis
    12. UNIT 10.11 Image Processing and 2-D Morphometry
    13. UNIT 10.12 Dial-In Flow Cytometry Data Analysis
    14. UNIT 10.13 The Application of Data Mining to Flow Cytometry
    15. Unit 10.14 Intensity Calibration and Flat-Field Correction for Fluorescence Microscopes
    16. UNIT 10.15 A Software Method for Color Compensation
    17. UNIT 10.16 Alternatives to Log-Scale Data Display
    18. UNIT 10.17 Web-Based Analysis and Publication of Flow Cytometry Experiments
    19. You have free access to this content
      UNIT 10.18 Preparing a Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) Compliant Manuscript Using the International Society for Advancement of Cytometry (ISAC) FCS File Repository (FlowRepository.org)
  14. Chapter 11 Microbiological Applications
    1. Introduction
    2. UNIT 11.1 Overview of Flow Cytometry and Microbiology
    3. UNIT 11.2 Flow Cytometry and Environmental Microbiology
    4. UNIT 11.3 Estimation of Microbial Viability Using Flow Cytometry
    5. UNIT 11.4 Sorting of Bacteria
    6. UNIT 11.5 Detection of Borreliacidal Antibodies by Flow Cytometry
    7. UNIT 11.6 Flow Cytometric Detection of Pathogenic E. coli in Food
    8. UNIT 11.7 Mycobacterium tuberculosis Susceptibility Testing by Flow Cytometry
    9. UNIT 11.8 Antibiotic Susceptibility Testing by Flow Cytometry
    10. UNIT 11.9 Determination of Bacterial Biomass from Flow Cytometric Measurements of Forward Light Scatter Intensity
    11. UNIT 11.10 Flow Cytometry of Yeasts
    12. UNIT 11.11 Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples
    13. UNIT 11.12 DNA/RNA Analysis of Phytoplankton by Flow Cytometry
    14. UNIT 11.13 Cell Cycle Analysis of Yeasts
    15. UNIT 11.14 Flow Cytometric Assessment of Drug Susceptibility in Leishmania infantum Promastigotes
    16. UNIT 11.15 Resolution of Viable and Membrane-Compromised Free Bacteria in Aquatic Environments by Flow Cytometry
    17. UNIT 11.16 Functional Assays of Oxidative Stress Using Genetically Engineered Escherichia coli Strains
    18. UNIT 11.17 Labeling of Bacterial Pathogens for Flow Cytometric Detection and Enumeration
    19. UNIT 11.18 Detection of Extracellular Phosphatase Activity of Heterotrophic Prokaryotes at the Single-Cell Level by Flow Cytometry
    20. UNIT 11.19 Life Cycle Analysis of Unicellular Algae
    21. You have free access to this content
      UNIT 11.20 Cytometry in Malaria—A Practical Replacement for Microscopy?
  15. Chapter 12 Cellular and Molecular Imaging
    1. Introduction
    2. UNIT 12.1 Comparative Overview of Flow and Image Cytometry
    3. UNIT 12.2 Basics of Digital Microscopy
    4. UNIT 12.3 Modern Confocal Microscopy
    5. UNIT 12.4 Time-Lapse Microscopy Approaches to Track Cell Cycle and Lineage Progression at the Single-Cell Level
    6. UNIT 12.5 Three-Dimensional Visualization of Blood and Lymphatic Vasculature in Tissue Whole Mounts Using Confocal Microscopy
    7. UNIT 12.6 Quantitative Fluorescence In Situ Hybridization (QFISH) of Telomere Lengths in Tissue and Cells
    8. UNIT 12.7 Detecting Protein–Protein Interactions with CFP-YFP FRET by Acceptor Photobleaching
    9. UNIT 12.8 Measuring FRET in Flow Cytometry and Microscopy
    10. UNIT 12.9 Live-Animal Imaging of Renal Function by Multiphoton Microscopy
    11. UNIT 12.10 Detecting Protein-Protein Interactions In Vivo with FRET using Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM)
    12. UNIT 12.11 Confocal Imaging of Cell Division
    13. UNIT 12.12 From In Vitro to In Vivo: Imaging from the Single Cell to the Whole Organism
    14. UNIT 12.13 Use of Spectral Fluorescence Resonance Energy Transfer to Detect Nitric Oxide-Based Signaling Events in Isolated Perfused Lung
    15. UNIT 12.14 Flow Cytometric FRET Analysis of ErbB Receptor Tyrosine Kinase Interaction
    16. UNIT 12.15 Cryosectioning
    17. UNIT 12.16 Immunohistochemistry
    18. UNIT 12.17 Simultaneous Optical Mapping of Intracellular Free Calcium and Action Potentials from Langendorff Perfused Hearts
    19. UNIT 12.18 Total Internal Reflection Fluorescence (TIRF) Microscopy
    20. UNIT 12.19 3D Deconvolution Microscopy
    21. UNIT 12.20 Approaches to Spectral Imaging Hardware
    22. UNIT 12.21 From Image to Data Using Common Image-Processing Techniques
    23. UNIT 12.22 Setting Up and Running an Advanced Light Microscopy and Imaging Facility
    24. UNIT 12.23 Photoactivation and Imaging of Optical Highlighter Fluorescent Proteins
    25. UNIT 12.24 Practical Methods for Molecular In Vivo Optical Imaging
    26. UNIT 12.25 Characterization of Surface FAS—Quantitative Morphological Analysis Using Quantitative Imaging Cytometry
    27. UNIT 12.26 Two-Photon Imaging of the Immune System
    28. UNIT 12.27 Near-Infrared Molecular Probes for In Vivo Imaging
    29. UNIT 12.28 Live Imaging of the Lung
    30. UNIT 12.29 Total Internal Reflection Fluorescence (TIRF) Microscopy Illuminator for Improved Imaging of Cell Surface Events
    31. UNIT 12.30 A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part I: BacMam Labeling and Reagents for Vesicular Structures
    32. UNIT 12.31 A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part II: Reagents for Non-Vesicular Organelles
    33. UNIT 12.32 A Review of Reagents for Fluorescence Microscopy of Cellular Compartments and Structures, Part III: Reagents for Actin, Tubulin, Cellular Membranes, and Whole Cell and Cytoplasm
    34. Unit 12.33 A Rapid and Sensitive Automated Image-Based Approach for In Vitro and In Vivo Characterization of Cell Morphology and Quantification of Cell Number and Neurite Architecture
    35. UNIT 12.34 Imaging Autophagy
    36. UNIT 12.35 The Application of KillerRed for Acute Protein Inactivation in Living Cells
  16. Chapter 13 Multiplexed and Microparticle-Based Analyses
    1. Introduction
    2. UNIT 13.1 Multiplexed Microsphere-Based Flow Cytometric Immunoassays
    3. UNIT 13.2 Microsphere Surface Protein Determination Using Flow Cytometry
    4. UNIT 13.3 Use of Microsphere-Supported Phospholipid Membranes for Analysis of Protein-Lipid Interactions
    5. UNIT 13.4 Multiplexed SNP Genotyping Using Primer Single-Base Extension (SBE) and Microsphere Arrays
    6. UNIT 13.5 BeadCons: Detection of Nucleic Acid Sequences by Flow Cytometry
    7. UNIT 13.6 Characterization of Nuclear Receptor Ligands by Multiplexed Peptide Interactions
    8. UNIT 13.7 Detection of Gene Fusions in Acute Leukemia Using Bead Microarrays
    9. UNIT 13.8 Reagents and Instruments for Multiplexed Analysis Using Microparticles
    10. UNIT 13.9 Multiplexed Detection of Fungal Nucleic Acid Signatures
    11. UNIT 13.10 Multiplexed Analysis of Peptide Antigen-Specific Antibodies
    12. UNIT 13.11 Use of Flow Cytometric Methods to Quantify Protein-Protein Interactions
    13. UNIT 13.12 Microsphere-Based Flow Cytometry Protease Assays for Use in Protease Activity Detection and High-Throughput Screening
    14. UNIT 13.13 Application of the PrimRglo Assay Chemistry to Multiplexed Bead Assays
  17. Appendix 1 Abbreviations and Useful Data
    1. APPENDIX 1A Abbreviations Used in this Manual
    2. APPENDIX 1B Common Conversion Factors
  18. Appendix 2 Stock Solutions, Equipment, and Laboratory Guidelines
    1. APPENDIX 2A Common Stock Solutions, Buffers, and Media
  19. Appendix 3 Commonly Used Techniques
    1. APPENDIX 3A Cell Counting
    2. APPENDIX 3B Techniques for Mammalian Cell Tissue Culture
    3. APPENDIX 3C Diagnosis and Treatment of Mycoplasma-Contaminated Cell Cultures
    4. APPENDIX 3D Wright-Giemsa and Nonspecific Esterase Staining of Cells
    5. APPENDIX 3E Techniques for Bacterial Cell Culture: Media Preparation and Bacteriological Tools
    6. APPENDIX 3F Growing Bacteria in Liquid Media
    7. APPENDIX 3G Growing Bacteria on Solid Media
    8. APPENDIX 3H Importing Biological Materials
    9. APPENDIX 3I Production of Polyclonal Antisera
    10. APPENDIX 3J Production of Monoclonal Antibodies
    11. APPENDIX 3K Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization

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