Spectral and Imaging Cytometry: Methods and Protocols

Dedication
Preface
References
Contents
Contributors
Part I: Spectral Flow Cytometry
Chapter 1: Development of Spectral Imaging Cytometry
1 Introduction
2 Development of Spectral Flow Cytometry
3 Current Spectral Cytometry Instruments
4 Advances and Limitations of Spectral Flow Cytometry
5 Development of Spectral Unmixing Algorithms
6 Spectral Unmixing Problems
7 Comparison of Spectral Unmixing and Spectral Compensation
8 Comparison of Spectral Cytometry and Mass Cytometry
9 Differences and Similarities Between Spectral and Conventional Flow Cytometry
10 Applications of Spectral FCM
11 Current Applications: Multi-parametric Spectral Cytometry
12 Two Major Types of Spectral FCM Analysis: Virtual Filtering and Spectral Unmixing
13 Conclusions
References
Chapter 2: Using Virtual Filtering Approach to Discriminate Microalgae by Spectral Flow Cytometer
1 Introduction
2 Materials
2.1 Instrumentation and Accessories
2.2 List of Microalgae Cell Cultures
2.3 Reagents
3 Methods
3.1 Cultivation of Algae Cultures
3.2 Spectrocytofluorimetric Acquisition of Microalgal Samples
3.3 Spectral Analysis of Algal Cell Cultures for ID 7000
3.4 Analysis of Microalgal Samples on a Spectral Flow Cytometer (Example with Cultures Acquired with ID7000 Spectral Flow Cyto...
3.5 Virtual Filtering (Example with Cultures Acquired with ID6800 Spectral Flow Cytometer)
3.6 Analysis of Microalgal Environmental Populations
3.7 Autofluorescence Finder
4 Notes
References
Part II: Imaging Flow Cytometry: Imagestream Systems
Chapter 3: Imaging Flow Cytometric Analysis of Primary Bone Marrow Erythroblastic Islands
1 Introduction
2 Materials
2.1 Collection of Bone Marrow
2.2 Staining and Preparation of EBIs
2.3 IFC Sample Acquisition and Analysis
3 Methods
3.1 Isolation of Bone Marrow
3.2 Fixation, Staining, and Preparation for IFC
3.3 IFC Setup and Acquisition
3.4 IFC Gating Strategy for Gated Erythroid-Macrophage Aggregates (GEMAs)
3.5 IFC Manual Screening of GEMAs for Potential EBIs
3.6 IFC Manual Screening of Potential EBIs for True EBIs
3.7 IFC Quantification of True EBIs
4 Notes
References
Chapter 4: Imaging Flow Cytometry of Legionella-Containing Vacuoles in Intact and Homogenized Wild-Type and Mutant Dictyosteli...
1 Introduction
1.1 Virulence of Legionella pneumophila and Formation of the Pathogen Vacuole
1.2 LCV Composition in D. discoideum Parental and Mutant Strains
1.3 Imaging Flow Cytometry Analysis of Pathogen Vacuole Formation
2 Materials
2.1 Legionella Pneumophila Strains
2.2 Culture Medium for L. Pneumophila Strains
2.3 Instrumentation
2.4 Dictyostelium Discoideum Strains
2.5 Dictyostelium Discoideum Medium
2.6 Additional Instrumentation and Stock Solutions
2.7 Experimental Infections. Intact Host Cells
2.8 Experimental Infections. Homogenized Host Cells
2.9 Imaging Flow Cytometry
3 Methods
3.1 Culture of L. Pneumophila
3.2 Culture of D. Discoideum
3.3 Experimental Infections for the Analysis of Intact Host Cells
3.4 Experimental Infections for the Analysis of Homogenized Host Cells
3.5 Acquisition of Imaging Flow Cytometry Data on Intact Host Cells
3.6 Acquisition of Imaging Flow Cytometry Data from Homogenized Host Cells
3.7 Analysis of Imaging Flow Cytometry Data of Intact Host Cells
3.8 Imaging Flow Cytometry of Homogenized Host Cells
4 Notes
References
Chapter 5: Imaging Flow Cytometry of Multi-Nuclearity
1 Introduction
2 Materials
2.1 Laboratory Instrumentation and Accessories
2.2 Reagents
3 Methods
3.1 Cancer Cell Line A549 Culturing
3.2 Growing A549 Cellular Monolayer with Taxol
3.3 Growing A549 Cellular Monolayer with Cytochalasin D
3.4 Preparation of Multi-Nucleated Cellular Suspension after Taxol or Cytochalasin D Treatment for Analysis on Imagestream X M...
3.5 Analysis of Micronuclei in Multi-Nucleated Cells under Microscope
3.6 Preparation Cells for Imaging Flow Cytometry Analysis
3.7 Starting and Running Imagestream X Mark II
3.8 Evaluation of Multi-Nuclear Cells Using Machine-Learning Module
4 Notes
References
Chapter 6: The Imaging Flow Cytometry-Based Cytokinesis-Block MicroNucleus (CBMN) Assay
1 Introduction
2 IFC CBMN Method
3 Materials
3.1 Lab Equipment
3.2 Reagents
4 Methods
4.1 Sample Preparation and Culture
4.2 Cell Lysing and Fixation
4.3 Staining
4.4 Data Acquisition and Analysis
5 Notes
References
Chapter 7: High-Throughput γ-H2AX Assay Using Imaging Flow Cytometry
1 Introduction
2 Materials
2.1 Lab Equipment
2.2 Reagents
3 Methods
3.1 Sample Preparation and Culture
3.2 Lysis and Fixation
3.3 Cell Staining
3.4 Data Acquisition and Analysis
4 Notes
References
Chapter 8: Label-Free Identification of Persistent Particles in Association with Primary Immune Cells by Imaging Flow Cytometry
1 Introduction
2 Materials
3 Methods
3.1 Blood Collection and Particle Incubation
3.2 Red Blood Cell Lysis
3.3 Dead Cell Staining
3.4 Phenotypic Marker Surface Staining
3.5 Acquisition Using an ImageStreamX Platform. Instrument Set up
3.6 Acquisition Using an ImageStreamX Platform. Spectral Overlap Compensation
3.6.1 Data Analysis. Initial Gating for Single, Focused, Cells Positive for Phenotypic Markers
3.6.2 Data Analysis. Measuring Particle Association with Cells with Darkfield Image Analysis
3.7 Measurement of Particle Cell Association Using Brightfield Images
4 Notes
References
Chapter 9: ``Immuno-FlowFISH´´: Applications for Chronic Lymphocytic Leukemia
1 Introduction
2 Materials
2.1 Buffers
2.2 Cell Preparation
2.3 Immuno-flowFISH Detection Panels for Chronic Lymphocytic Leukemia (CLL)
2.4 Immunophenotyping, Cell Fixation and Permeabilization
2.5 Acid Denaturation and FISH
2.6 Additional Materials
2.7 Instrumentation
3 Methods
3.1 Preparation of Leucocytes
3.2 Immunophenotyping, Cell Fixation, and Permeabilization
3.3 Acid Denaturation and Probe Hybridization
3.4 Post-hybridization Wash and DNA Stain
3.5 AMNIS ISX MarkII Instrument Setup
3.6 Sample Acquisition
3.7 IDEAS Data Analysis
4 Notes
References
Chapter 10: Quantifying Golgi Apparatus Fragmentation Using Imaging Flow Cytometry
1 Introduction
2 Materials
2.1 Cell Culture and Stimulation
2.2 Cell Collection and Fixation
2.3 Immuno-fluorescence Labeling
2.4 Imaging Flow Cytometry and Image Analysis
3 Methods
3.1 Cell Culture and Stimulation
3.2 Cell Collection and Fixation
3.3 Immuno-fluorescence Labeling
3.4 Image Acquisition
3.5 Image Analysis
4 Expected Results
5 Notes
References
Chapter 11: Flow Imaging of the Inflammasome: Evaluating ASC Speck Characteristics and Caspase-1 Activity
1 Introduction
2 Materials
2.1 Cells
2.2 Cell Culture
2.3 Reagents
2.4 Instrument and Software
2.5 Expression Plasmids and DNA Transfection
3 Methods (Fig. 1)
3.1 Inflammasome Reconstitution and Activation
3.2 THP1 Cells
3.3 Immortalized BMDMs (iBMDMs): Wild-type and Caspase-1/11-/-
3.4 Primary Human Monocytes (See Note 4)
3.5 Preparation of Samples for Acquisition by the Amnis ImageStream X
3.6 ImageStream X Acquisition Parameters: (See Note 7)
3.7 Compensation and Data Analysis Template Generation on IDEAS
4 Notes
References
Chapter 12: Quantitative Analysis of Latex Beads Phagocytosis by Human Macrophages Using Imaging Flow Cytometry with Extended ...
1 Introduction
2 Materials
2.1 Reagents
2.2 Laboratory Equipment and Accessories
2.3 Cell Culture
3 Methods
3.1 Cells and Macrophage Differentiation Protocol
3.2 Phagocytosis Assay Technique
3.3 Data Acquisition and Analysis
4 Notes
References
Part III: Imaging Flow Cytometry: FlowCam
Chapter 13: FlowCam 8400 and FlowCam Cyano Phytoplankton Classification and Viability Staining by Imaging Flow Cytometry
1 Introduction
2 Materials
2.1 Algal Cultures and Natural Samples Collection
2.2 Instrumentation Accessories and Software
3 Methods
3.1 How to Qualify the Laser and PMT Performance in FlowCam 8400S Using Fluorescent Beads
3.2 How to Distinguish Cyanobacteria with Phycocyanin and Chlorophyll Fluorescence from Phytoplankton Containing Only Chloroph...
3.3 Fluorescent Filters Can Discriminate Nuisance Cyanobacteria from Other Algae and Detritus in Natural Freshwater Samples
3.4 Discrimination of Algal Types by Visual Determination of Filter Boundary Using the ``View Window´´
3.5 Discrimination of Algal Types by Using the Ch2:Ch1 log ratio-Based Filter
3.6 Using Libraries to Help Sort Algal Sample Runs
3.7 Viability Staining with GRS Dye
3.8 Viability Staining with Fluorescein Diacetate Dye
3.9 Viability Staining with RFS Dye
3.10 Viability Staining with FarRFS Dye
4 Notes
References
Chapter 14: Optimizing FlowCam Imaging Flow Cytometry Operation for Classification and Quantification of Microcystis Morphospe...
1 Introduction
2 Materials
2.1 Instrumentation and Accessories
2.2 Sample Preparation
3 Methods
3.1 Sample Processing
3.2 Data Classification
4 Notes
References
Index