Flow Cytometry Protocols (Methods in Molecular Biology, 2779)

Dedication
Preface
Contents
Contributors
Chapter 1: Flow Cytometry: The Glass Is Half Full
	1 Introduction
	References
Chapter 2: Basic Principles of Flow Cytometer Operation: The Make your Own Flow Cytometer
	1 Introduction
	2 Materials
	3 Methods
		3.1 Instrument Assembly
			3.1.1 Lasers and Their Optics
			3.1.2 Cell Delivery: The Flow Cell or Cuvette
			3.1.3 The Instrument´s Fluidics
			3.1.4 Turning on the Sample Stream
			3.1.5 Light Collection, Dichroic Mirrors, and Filters
			3.1.6 Side Scatter and Fluorescence
			3.1.7 Forward Scatter
			3.1.8 Electronics
		3.2 Data Acquisition and Analysis
		3.3 The Fully Assembled Flow Cytometer: Optics, Fluidics, Electronics, and Software
	References
Chapter 3: Laser Sources for Traditional and Spectral Flow Cytometry
	1 Introduction
	2 Laser and Cytometer Characteristics
		2.1 How Many Lasers and which Wavelengths
		2.2 Traditional Versus Spectral Flow Cytometry
		2.3 Instrument Characteristics
			2.3.1 Cuvette Versus Stream-in-Air
			2.3.2 Spatially Separated Versus Colinear Laser Systems
			2.3.3 Fiber Optic-Coupled Versus Free-Space Lasers
			2.3.4 Post-Laser Beam Modification Optics in the Cytometer Design
		2.4 Laser Characteristics
			2.4.1 The Mechanism of Operation
			2.4.2 The Power Level
			2.4.3 Continuous Wave (CW) Versus Quasi-CW
			2.4.4 Laser Beam Characteristics and Beam Quality
			2.4.5 Laser Coherence and Wavelength Stability
			2.4.6 Operational Lifetime
			2.4.7 The Form Factor
	3 Laser Wavelengths
		3.1 Cyan 488 Nm
		3.2 Red Lasers
		3.3 Violet Lasers
		3.4 Green-to-Yellow Lasers
		3.5 Ultraviolet Lasers
		3.6 Blue Lasers
		3.7 Orange Lasers
		3.8 Near-Infrared (NIR) Lasers
		3.9 Deep Ultraviolet (DUV) Lasers
		3.10 Unusual Laser Wavelengths
		3.11 Multi-Wavelength Lasers
		3.12 Merge Modules or Laser Engines
	4 Laser Combinations
		4.1 Combinations for Conventional Cytometers
		4.2 Combinations for Spectral Cytometers
	References
Chapter 4: How to Measure ``Spillover Spread´´
	1 Introduction
	2 Materials
	3 Methods (See Note 2)
		3.1 The  SSM
			3.1.1 Sample Preparation
			3.1.2 Setting up the Flow Cytometer
			3.1.3 Calculating the  SSM
		3.2 The  SQI
			3.2.1 Sample Preparation and Setting up the Flow Cytometer
			3.2.2 Calculating the  SQI
	4 Notes
	References
Chapter 5: Comprehensive Immunophenotyping by Polychromatic Cytometry
	1 Introduction
	2 Materials
		2.1 Instruments and Supplies
		2.2 Reagents
		2.3 Prepared Solutions
		2.4 Samples
	3 Methods
		3.1 Panel Design
		3.2 Titration of Antibodies
		3.3 Preparation of PBMCs
		3.4 PBMC Staining
		3.5 Single-Stained Controls
		3.6 Data Analysis
	4 Notes
	References
Chapter 6: Panel Design and Optimization for Full Spectrum Flow Cytometry
	1 Introduction
	2 Materials
		2.1 Panel Design
		2.2 Panel Optimization
		2.3 Autofluorescence-Evaluation and Strategies
		2.4 Longitudinal Studies
	3 Methods
		3.1 Panel Design (Fig. 2)
			3.1.1 Determine the Experimental Hypothesis and Biological Context for Optimal Panel Design
			3.1.2 Understanding Marker Expression and Density
			3.1.3 Fluorophore Selection
			3.1.4 Assign Fluorophores to Markers Based on Fluorophore Uniqueness, Brightness, and Spread Properties
			3.1.5 Review the Theoretical Panel Design
		3.2 Panel Optimization (Fig. 3)
			3.2.1 Antibody Titrations
			3.2.2 Preparation and Evaluation of Optimal Single-Stain (SS) Controls
			3.2.3 Unmixing Accuracy
			3.2.4 Evaluation of Marker Resolution
			3.2.5 Assessment of Data Quality Using Expert Gating and Dimensionality Reduction Algorithms
		3.3 Strategies for Autofluorescence Characterization (Fig. 4)
			3.3.1 Evaluate Accuracy of Unmixing and Determine Whether Additional AF Tags Are Required
			3.3.2 For Complex Heterogeneous Samples: Identify the Unique AF Signatures Present
			3.3.3 Identify Which AF Signatures Are Homogeneous and Unique
			3.3.4 Unmix Multicolor Samples with New AF  Tags
			3.3.5 Evaluate Unmixing with New  Tags
		3.4 Longitudinal Studies (Fig. 8)
			3.4.1 Generate Preserved Representative Reference Material (RRM)
			3.4.2 Setup Instrument Control
			3.4.3 Stain and Acquire Samples
			3.4.4 Evaluate Batch Effects and Normalize Data
	4 Notes
	References
Chapter 7: Practicalities of Cell Sorting
	1 Introduction
	2 Cell Analysis
	3 Drop-Based Sorting
	4 Sorting Principle of Drop-Based Sorters
	5 How Is the Drop Delay Calculated?
	6 Considerations for a Successful Cell Sorting Experiment
	7 Sort Modes
	8 How Long Does It Take to Sort Your Target Population
	9 Bio-containment
	10 Quality Control (QC)
	11 Cell Preparation and Sort Buffers
	12 Cell Preparation for Cell Sorting
		12.1 Protocol: PBMC (Peripheral Blood Mononuclear Cells)
		12.2 Protocol: Adherent Cells from Cell Line
	13 Cleaning
		13.1 A Generic Cell Sorter Cleaning Protocol Would Be
		13.2 Cleaning Before a Sort for Cell Culture (Aseptic Sorting)
		13.3 Cleaning Before a Sort for RNA Purification
		13.4 Cleaning Between Users or Before Running the Instruments Shutdown Procedure
	14 Example Protocol for Sorting GFP Transfected Cell Lines
	15 Evaluation of Performance When Sorting into Plates and PCR Tubes
	References
Chapter 8: Image-Enabled Cell Sorting Using the BD CellView Technology
	1 Introduction
		1.1 The Rationale for Using Image-Enabled Cell Sorting
		1.2 BD CellView Image-Enabled Cell Sorting Technology
		1.3 Sort-Enabled Image Analysis Parameters
		1.4 Example of a Novel Application: Measuring and Sorting Out Chemically Induced Golgi Fragmentation in GalNac-GFP HeLa Cells
	2 Conclusion
	References
Chapter 9: Monitoring Cell Proliferation by Dye Dilution: Considerations for Panel Design
	1 Introduction
	2 Materials
		2.1 Cell Isolation and Cell Culture
		2.2 Antibodies and Immunophenotyping Reagents
		2.3 Flow Cytometry Reagents
		2.4 Cell and Proliferation Tracking Dyes
		2.5 Flow Cytometers, Other Equipment, and Data Analysis Software
	3 Methods
		3.1 Cell Line and hPBMC Labeling with Protein Dyes (CTV, CFSE, CTFR, or CYY)
		3.2 Cell Line and hPBMC Labeling with Membrane Dyes (PKH26, PKH67, or CVC)
		3.3 Evaluating Linearity of Dye Dilution
			3.3.1 Relative Growth Rate of Stained vs. Unstained Cells Using Counting Beads (Nonvolumetric Cytometer)
			3.3.2 Relative and Absolute Cell Growth Rate Determination in Co-cultures (Volumetric Cytometer)
			3.3.3 Assessing Linearity of Dye Dilution and Relative Dilution Rates
		3.4 Biological Considerations for Cell Proliferation Modeling
		3.5 Spectral Characterization
			3.5.1 Spectral Overlap and Cross-Laser Excitation
			3.5.2 Assessing the Impact of Fluorochrome Choice on Resolution of Antibody Positive and Negative Cells
		3.6 Evaluating the Effect of Tracking Dye Labeling on Cellular Function(s)
			3.6.1 Preparation of Monocyte Depleted Lymphocytes (See Note 41)
			3.6.2 Isolation of Treg, Teff, and Accessory Cells by Flow Cytometry and Sorting (See Note 45)
			3.6.3 Proliferation Protocol
			3.6.4 Flow Cytometric Data Acquisition and Analysis
			3.6.5 Calculation of Proliferative Fraction and Proliferative Index
	4 Notes
	References
Chapter 10: Multiparametric Analysis of Apoptosis by Flow Cytometry
	1 Introduction
	2 Materials
		2.1 Fluorogenic Caspase Assays
		2.2 Annexin V
		2.3 DNA-Binding Dyes
		2.4 Covalent Viability Probes
		2.5 Anti-active Caspase 3 Antibody
		2.6 Combinations of Fluorochromes
			2.6.1 Single Cyan 488 nm Laser Instruments
			2.6.2 Dual Cyan/Red Laser-Equipped Instruments
			2.6.3 Triple Cyan/Red Laser/Violet Laser-Equipped Instruments
			2.6.4 Other Lasers
		2.7 Buffers and Labeling Reagents
	3 Methods
		3.1 Preparation of Cells
		3.2 Fluorogenic Caspase Substrate Labeling
			3.2.1 PhiPhiLux G1D2 Caspase 3/7 Substrate
			3.2.2 FLICA
			3.2.3 CellEvent Green or NucView 488
		3.3 Annexin V Labeling
		3.4 DNA-Binding Dye Labeling
		3.5 Covalent Viability Probe Labeling
		3.6 Fixable Assays Using FLICA and Covalent Viability Probes
		3.7 Immunolabeling for Active Caspase 3
		3.8 Flow Cytometric Analysis
		3.9 Data Acquisition and Analysis
			3.9.1 Scatter Gating
			3.9.2 Annexin V Binding and DNA-Binding Dye Exclusion
			3.9.3 Covalent Viability Probes
			3.9.4 Fluorogenic Caspase Substrates
		3.10 Sample Results for Multiparametric Apoptosis Assays
			3.10.1 PhiPhiLux, Annexin V, and DNA-Binding Dye
			3.10.2 FLICA, Annexin V, and DNA-Binding Dye
			3.10.3 CellEvent Green, Annexin V, and DNA-Binding Dye
			3.10.4 FLICA and Covalent Viability Probe
			3.10.5 Immunolabeling for Caspase 3
		3.11 Conclusion
	4 Notes
	References
Chapter 11: Quantitative and Standardized Pseudovirus Neutralization Assay for COVID-19
	1 Introduction
	2 Materials
		2.1 Raw Materials and Reagents
		2.2 Equipment
	3 Methods
		3.1 Splitting Cells from a T75 Culture Flask
		3.2 Plating Cells in a Flat-Bottom 12-Well TC Plate for Cytometer Compensation
		3.3 Plating Cells for a 96-Well Assay
		3.4 Neutralization of Pseudovirus with Serum Sample
		3.5 Flow Cytometer Compensation
		3.6 Detection of GFP-Pseudovirus Infection and Calculation of 50% Neutralization (NT50)
	4 Notes
	References
Chapter 12: CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting
	1 Introduction
	2 Materials
		2.1 FP-RMS Cell Lines
		2.2 Transfection Reagents
		2.3 Single-Cell Cloning of CRISPR-Cas9 HiBiT Knock-In Cells
		2.4 Validation of CRISPR-Cas9 HiBiT Knock-In Cells
	3 Methods
		3.1 Plasmid Encoding Cas9 and sgRNAs
		3.2 Plasmid Encoding DNA Construct for Homology Directed Repair
		3.3 Generation of CRISPR-Cas9 HiBiT Knock-In Cell Lines
		3.4 Validation of CRISPR-Cas9 HiBiT Knock-In Cells
	4 Notes
	References
Chapter 13: Co-staining with Fluorescent Antibodies and Antibody-Derived Tags for Cell Sorting Prior to CITE-Seq
	1 Introduction
	2 Materials
		2.1 Flow Proxy Assay
		2.2 Co-staining Cells with Fluorescent Antibodies and  ADTs
		2.3 Cell Sorting
		2.4 Single-Cell CITE-Seq Using the BD Rhapsody System
	3 Methods
		3.1 (Optional) Flow Proxy Assay to Evaluate the Binding of Fluorescent Antibody and ADT to the Same Protein Marker
		3.2 Co-staining PBMCs with a Fluorescent Panel and an ADT Panel (See Note 8)
		3.3 Fluorescence-Activated Cell Sorting to Isolate Cell Population of Interest
		3.4 Single-Cell Capture Using the BD Rhapsody Single-Cell Analysis System
	4 Notes
	References
Chapter 14: Discovery of Live Cell Selective Fluorescent Probes and Elucidation of Their Mechanisms: Case Study of B Cell Sele...
	1 Introduction
	2 Materials
		2.1 Reagents and Cells
		2.2 Equipment and Supplies
	3 Methods
		3.1 Screening for a B Lymphocyte Selective Carbon Length Fluorescent Compound
			3.1.1 Preparation of Splenocytes
			3.1.2 Staining with B Cell Antibody (CD45R/B220) or B Cell-Specific Fluorescent Probe (CDgB)
			3.1.3 Staining with Small-Molecule Fluorescent Compound
			3.1.4 Acquisition of FCS Files Using Flow Cytometry
			3.1.5 Establishment of Standards for Separation Ratio Analysis
			3.1.6 SI (Stain Index) Analysis
			3.1.7 Comprehensive Analysis of Screening Results
		3.2 Analysis of CDgB Staining Mechanism Using Small Unilamellar Vesicles (SUVs) Cell Mimicking Model
			3.2.1 Preparation of SUVs
			3.2.2 Screening of SUVs with CDgB
	4 Notes
	References
Chapter 15: Fluorescence Lifetime Measurements and Analyses: Protocols Using Flow Cytometry and High-Throughput Microscopy
	List of Variables
	1 Introduction
	2 Fluorescence Lifetime Theory and Praxis
		2.1 Theory
		2.2 Praxis
		2.3 Phasor Analyses
	3 Instrumentation for Lifetime Measurements: Cytometers and High-Throughput Imagers
		3.1 Time-Resolved Flow Cytometry
		3.2 High-Throughput FLIM (ht-FLIM)
	4 Applications and Protocols
		4.1 Exogenous Fluorophores and Microspheres
		4.2 Endogenous Fluorophores
		4.3 Fluorescent Proteins
		4.4 Förster Resonance Energy Transfer
	5 Conclusion
	References
Chapter 16: Using Artificial Intelligence to Interpret Clinical Flow Cytometry Datasets for Automated Disease Diagnosis and/or...
	1 Introduction
		1.1 Machine Learning and Representation Learning
		1.2 Objective of Developing an Automated Flow Analysis Platform via Representation Learning
	2 Materials
		2.1 Prepare the Training Dataset Using Real-World  Data
		2.2 Training Dataset for Each Use  Case
	3 Methods
		3.1 Flow Cytometry Tests and Interpretation
			3.1.1 MFC Data Collection
		3.2 Classification Model Development
		3.3 Algorithm Performance Assessment
		3.4 Misclassification Cause Analysis
		3.5 Feature Selection Analysis
		3.6 Discussion
	4 Notes
	References
Chapter 17: Approaching Mass Cytometry Translational Studies by Experimental and Data Curation Settings
	1 Introduction
	2 Principles of Mass Cytometry
		2.1 Basic CyTOF Experiment Workflow
		2.2 Improvements in CyTOF Instrumentation
		2.3 Comparison of CyTOF Assays Across Multiple Instruments
	3 Reducing Sample Variation in the Context of CyTOF Experiments
		3.1 Importance of the Reagent´s Quality and Sample Calculation
		3.2 Sample Collection and Storage
		3.3 Intra and Interexperimental Variation Control
			3.3.1 One Common Antibody Mastermix
			3.3.2 Barcoding
			3.3.3 Technical Replicates
			3.3.4 Calibration Beads
	4 Data Curation Tools
		4.1 Data Transformation
		4.2 Data Compensation
		4.3 Flow Rate and Signal Quality Check
		4.4 Data Debarcoding
		4.5 Staining Quality Check (AOF)
		4.6 Data Normalization
			4.6.1 Bead-Based Normalization
			4.6.2 Normalization Based in Technical Replicates
			4.6.3 Normalization Methods Without Technical Replicates
		4.7 Pregating
	5 Conclusions
	References
Chapter 18: CyTOF Intracellular Cytokine Assays for Antigen-Specific T Cells
	1 Introduction
	2 Materials
		2.1 Reagents (See Note 1)
		2.2 Equipment and Supplies
	3 Methods
		3.1 Thawing of   PBMC
		3.2 Cell Activation
			3.2.1 Without Enrichment
			3.2.2 With Enrichment
		3.3 Viability Dye and Cell-Surface Staining
		3.4 Fixation and Permeabilization
		3.5 Intracellular Staining
	4 Notes
	References
Chapter 19: Imaging Mass Cytometry for In Situ Immune Profiling
	1 Introduction
	2 Materials
		2.1 Reagents for Basic IMC Protocol
		2.2 Supplies and Instruments for Basic IMC Protocol
		2.3 (Optional) Additional Materials for IMC Protocol with RNAscope
			2.3.1 Reagents
			2.3.2 Supplies and Instruments
	3 Methods
		3.1 Designing the Staining Panel
		3.2 Preparation of Tissue Sample
		3.3 Section Staining
			3.3.1 Histological Slide Preparation
			3.3.2 (Optional) Staining for RNA Targets
			3.3.3 Staining for Protein Markers
			3.3.4 Staining with DNA Intercalator
		3.4 Hyperion Acquisition
		3.5 Image Visualization
	4 Notes
	References
Chapter 20: Cytometry in High-Containment Laboratories
	1 Introduction
	2 Emerging Infectious Diseases: A Public Health Perspective
	3 Emerging Infectious Diseases: Biosafety, Biosecurity, and High Containment
		3.1 Risk Groups, Biosafety Levels, and High Containment  Labs
		3.2 Global Expansion of High Containment Laboratories
	4 Framework for Integrating New Technologies into High Containment
		4.1 Survey of Technologies
		4.2 Perform the Risk Assessment
		4.3 Develop Standard Operating Procedures
		4.4 Investigate at BSL-2
		4.5 Integration and Evaluation
		4.6 Continual Development and Training
	5 Cytometry Technologies, Applications, and Challenges
		5.1 Cytometry Technologies
			5.1.1 Cell Analysis and Sorting
			5.1.2 CyTOF
			5.1.3 Image-Based Cytometry
			5.1.4 Cytometric Bead Array
			5.1.5 Serum Proteomics
			5.1.6 Single-Cell Technologies
		5.2 High Containment Adaptations and Challenges
			5.2.1 Approaches to Sample Handling
			5.2.2 Sorting Procedures in High Containment
			5.2.3 Challenges with Cytometry in High Containment
	6 Future Outlooks and Applications
	7 Conclusion
	References
Index