Preface
Contents
Contributors
Part I: Purification and Phenotypic Characterization of B Cell Subsets with Breg Traits
Chapter 1: Purification and Characterization of Murine MZ and T2-MZP Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions for Cell Preparation
2.3 Flow Cytometry Analysis
2.4 Immunofluorescence
2.5 RNA Isolation and cDNA Synthesis for Repertoire Analysis
2.6 PCR Amplification, Product Purification, Subcloning, and Propagation in Bacteria
3 Methods
3.1 Tissue Collection for Immunohistochemistry
3.2 Cell Preparation
3.3 Staining Procedures for Flow cytometry Analysis
3.4 Staining Procedures for Cell Sorting
3.5 Slide Preparation and Storage
3.6 Confocal Microscopy Staining
3.7 RNA Isolation and cDNA Synthesis for Repertoire Analysis
3.8 PCR Amplification and Purification of Immunoglobulin Heavy Chains for Repertoire Analysis
3.9 Subcloning into TOPO Cloning/Sequencing Vectors
3.10 Bacterial Plasmid DNA Purification and Sequencing of Immunoglobulin Heavy Chains
3.11 Analysis of the Immunoglobulin Heavy Chain Repertoire
4 Notes
References
Chapter 2: Purification and Immune Phenotyping of B-1 Cells from Body Cavities of Mice
1 Introduction
2 Materials
2.1 Mice
2.2 General Buffers and Reagents
2.3 Equipment
2.4 Antibodies
3 Methods
3.1 Collection of Peritoneal cavity Cells (PerC)
3.2 Collection of Pleural Cavity Cells (PlerC)
3.3 Magnetic-Based Cell Isolation of B-1 Cells from Peritoneal and Pleural Cavities
3.4 B-1 Cell Purity Check Via Flow cytometry Analysis
3.5 FACS-Sorting of B-1a and B-1b Cells
3.6 Immune Phenotyping of B-1a and B-1b Cells from Body Cavities
4 Notes
References
Chapter 3: Purification and Immunophenotypic Characterization of Murine Plasma Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Consumables
2.3 Animals
2.4 Antibodies and Stimulation Agonists
2.5 Buffers and Media
2.6 Other Reagents
3 Methods
3.1 Isolation of Splenocytes
3.2 Identification and Immunophenotype Characterization of LAG-3+ Regulatory Plasma cells by Flow Cytometry
3.2.1 Surface Staining
3.2.2 Intracellular Staining of LAG-3+ Regulatory Plasma Cells
3.3 Purification of LAG-3+ Natural Plasma Cells
3.4 Stimulation of LAG-3+ Regulatory Plasma cells and Evaluation of Cytokine Production
3.5 Detection of Antibody Production in LAG-3+ Plasma cells by ELISPOT Assay
4 Notes
References
Chapter 4: Purification of Murine and Human IL-10-Producing B Cells from Different Anatomical Compartments
1 Introduction
2 Materials
2.1 General Equipment
2.2 Buffers, Solutions, and Media
2.3 Mice and Biological Material
2.4 Reagents for B Cell Isolation and Culture
2.5 Reagents for Cell Staining
3 Methods
3.1 Splenic Murine IL-10+ B Cell Isolation
3.1.1 Isolation of Splenic IL-10-Secreting B Cells from Wild-Type Mice
3.1.2 Isolation of Splenic GFP+ B Cells from IL-10 Reporter GFP Knock-in Tiger Mice
3.2 Isolation of Peritoneal IL-10+ B Cells
3.3 Isolation of Human IL-10+ B Cells from the Peripheral Blood
4 Notes
References
Chapter 5: Purification and Immunophenotypic Characterization of Human CD19+CD24hiCD38hi and CD19+CD24hiCD27+ B Cells
1 Introduction
2 Materials
2.1 General Equipment
2.2 Buffer and Reagents
2.3 Reagents for B Cell Culture and Flow cytometry Analysis
3 Methods
3.1 Isolation of Peripheral Blood Mononuclear Cells from Whole Human Blood
3.2 B-Cell Isolation from Total Peripheral Blood Mononuclear Cells
3.3 Preparation of B Cells for Isolation of CD24hiCD38hi and CD24hiCD27+ B Cells Using FACS
3.4 Culturing B Cells for Breg Expansion
3.5 Fluorescent Staining for the Analysis of Surface Markers and Intracellular Cytokines by Flow Cytometry
3.6 Guidelines for the Analysis of Stained Cells by Flow Cytometry
4 Notes
References
Part II: Mechanisms of Immune Suppression by B Cells
Chapter 6: Detection of IL-10 in Murine B Cells: In Vitro and In Vivo Techniques
1 Introduction
2 Materials
2.1 Equipment
2.2 Media and Buffers
2.3 Cytokines, Antibodies, and Other Reagents
2.4 Reagents for IL-10 mRNA Measurement
2.5 Reagents for B-Cell Isolation
2.6 Mice
3 Methods
3.1 B Cell Purification and CD19+CD1dhiCD5+ Cell Isolation
3.2 IL-10 mRNA Measurement
3.2.1 mRNA Extraction
3.2.2 Complementary DNA (cDNA) Preparation
3.2.3 Design and Control of Primers for IL-10 cDNA Amplification
3.2.4 Quantitative Real-Time PCR (qPCR)
3.3 Intracellular IL-10 Measurement by Flow Cytometry
3.4 Detection of Secreted IL-10 by ELISA
3.5 Measuring of IL-10 Secreting B Cells by ELISPOT Assay
3.6 IL-10 Detection by Immunofluorescence Microscopy
3.7 Immunofluorescence Microscopy for the Detection of the Anatomical Distribution of CD19 + CD1dhiCD5+ B Cells In Situ
4 Notes
References
Chapter 7: Detection and Quantification of Transforming Growth Factor-Ξ²1 Produced by Murine B Cells: Pros and Cons of Differen...
1 Introduction
2 Materials
2.1 B-Cell Isolation
2.2 B Cell Culture
2.3 Flow Cytometry
2.4 Elisa
2.5 Real-Time PCR
3 Methods
3.1 B-Cell Isolation
3.2 B-Cell Culture
3.3 Detection of TGF-Ξ²1 by Flow Cytometry
3.4 Measurement of TGF-Ξ²1 Level in Cell Supernatant
3.5 RNA Extraction and Real-Time PCR
4 Notes
References
Chapter 8: IL-35 Detection in B Cells at the mRNA and Protein Level
1 Introduction
2 Materials
2.1 Equipment
2.2 General Buffers and Reagents
2.3 Induction of the PDAC Mouse Model and Isolation of Splenic and Pancreatic Tumor-Infiltrating B Cells
2.4 Isolation of Human B Cells
2.5 In Vitro Culture of Murine and Human B Cells
2.6 ELISA-Based Detection of IL-35
2.7 RT-qPCR Based Detection of IL-35
2.8 Immunofluorescence
2.9 Flow Cytometry
3 Methods
3.1 Induction of Murine Pancreatic Tumors
3.2 Isolation of Tumor-Infiltrating Lymphocytes
3.2.1 Murine Pancreatic Tumors Harvest and Digestion
3.2.2 Isolation of Tumor-Infiltrating Immune Cells
3.3 Isolation of Splenocytes from Tumor-Bearing Mice
3.4 Detection of Murine IL-35-Producing Breg Cells
3.4.1 Cell Staining for Sorting of Breg Cells
3.4.2 In Vitro Culture and Stimulation of Sorted B Cells
3.5 Analysis and Quantification of IL-35 Production by B Cells
3.5.1 Intracellular Cytokine staining for the Detection of IL-35-Producing B Cells
3.5.2 Real-Time Quantitative PCR for the Detection of IL-35 Transcripts
3.5.3 Detection of Circulating Murine IL-35 by ELISA
3.6 Identification and Detection of IL-35-Producing Human B Cells
3.6.1 Isolation of PBMCs from Human Peripheral Blood
3.6.2 Isolation and Activation of Human Breg Cells
3.6.3 Detection of IL-35-Expressing Human B Cells through Flow Cytometry
3.6.4 Detection of IL-35 Expression by Immunofluorescence in Human Pancreatic cancer Lesions
4 Notes
References
Chapter 9: Characterization and Activation of Fas Ligand-Producing Mouse B Cells and Their Killer Exosomes
1 Introduction
2 Materials
2.1 General Equipment
2.2 Mice
2.3 Media and Liquid Reagents
2.4 Materials for B-Cell Culture and exosomes Purification
2.5 Western Blot Reagents
2.6 Flow Cytometry Reagents
2.7 Reagents for B-Cell Cytokines and FasL ELISA
2.8 Measurement of Peptide-Specific Killing of Mouse TH Cells by Killer B Cells
3 Methods
3.1 Isolation of Purified B Lymphocytes and Exosomes from Mice
3.1.1 Preparation of Single-Cell Suspensions from Mouse Spleen (See Note 2)
3.1.2 Isolation of Cells from Mouse Lung Tissue
3.1.3 Collection of Peritoneal Cells by Abdominal Lavage
3.1.4 Immunomagnetic Purification of Mouse B Lymphocytes
3.1.5 FACS Sorting FasL+ B Cells
3.1.6 Isolation of Exosomes from Cell-Free spleen Extracts
3.1.7 Isolation of Exosomes from Culture Supernatants
3.2 Expansion of Mouse FasL+ B Cells by In Vitro Cell Culture
3.2.1 Culture of Mouse CD40 Ligand-Transduced NIH3T3 Fibroblasts
3.2.2 Cell Culture Recommendations for the Expansion of FasL+ B Cells
3.3 Phenotypic Analysis of Killer B Cells
3.3.1 Flow Cytometric Characterization of FasL+ B Cells
3.3.2 Western Blot Analysis for Cellular FasL Expression
3.4 Phenotypic Analysis of FasL+ Exosomes
3.4.1 Western Blot for FasL Protein in Exosomes
3.4.2 FasL ELISA of Exosome Protein Extracts
3.4.3 Bead Capture of Exosomes for Flow Cytometric Analysis
3.5 Functional Assays
3.5.1 Measurement of IL-10 Production by FasL+ B Cells Using Intracellular Staining
3.5.2 Measurement of Peptide-Specific, FasL-Dependent Killing of Mouse TH Cells by Killer B Cells
4 Notes
References
Chapter 10: Characterization and Activity of TIM-1 and IL-10-Reporter Expressing Regulatory B Cells
1 Introduction
2 Materials
2.1 Mice
2.2 Laboratory Plasticware
2.3 Equipment
2.4 Buffers, Media, and General Solutions
2.5 Reagents and Kits
3 Methods
3.1 Identification and Characterization of TIM-1+ B Cells
3.1.1 Preparation of Splenocytes Suspensions and Cell Stimulation Protocol for IL-10 Induction
3.1.2 Cell Staining
3.2 Isolation of TIM-1+ B Cells
3.2.1 Isolation of CD19+B Cells
3.2.2 Cell Sorting of Tim-1+ B Cells
3.3 Functional Assay for TIM-1+ Breg Cells
3.4 Detection of IL-10+ Bregs Utilizing IL-10 Reporter Mice
3.4.1 Direct Identification of IL-10+ Bregs In Vivo from Secondary Lymphoid Organs Using IL-10 Reporter mice by Flow Cytometry
3.4.2 In Vivo Detection of IL-10+ B Cells in Splenic Cryosections
3.5 Examination of Breg In Vivo Interactions by Two-Photon Intravital Microscopy Using IL-10 GFP Reporter Mice
3.5.1 Intravital Imaging of IL-10+ B Cells in Freshly Cut spleen Sections
3.5.2 Preparation of T Cells for Intravital Imaging (Day -1 from Breg and DC Transfer)
3.5.3 Preparation of Dendritic cells for Intravital Imaging (Same Day as CD19+GFP+ B Cells Transfer)
3.5.4 Analysis of In Vivo Breg Interactions
4 Notes
References
Chapter 11: New Method for the Expansion of Highly Purified Human Regulatory Granzyme B-Expressing B Cells
1 Introduction
2 Materials
2.1 General Equipment
2.2 Peripheral Blood Mononuclear Cells (PBMCs) Isolation
2.3 B-Cell Enrichment
2.4 Reagents for B-Cell Culture
2.5 Reagents for GZMB+ B-Cell Staining
2.6 Evaluation of Suppressive Function of GZMB+ B Cells by B/T-Cell coculture Assays
3 Methods
3.1 PBMCs Isolation
3.2 B-Cell Enrichment by Immunomagnetic Separation
3.3 GZMB+ B-Cell Expansion and Staining
3.4 Evaluation of the Suppressive Function of GZMB+ B Cells by B/T-Cell coculture Assays
3.4.1 T-Cell Preparation
3.4.2 Set up of the B/T-Cell Coculture
3.4.3 Evaluation of the Suppression of T-Cell Proliferation by Flow Cytometry
4 Notes
References
Chapter 12: Characterization of the Cell Surface Phenotype and Regulatory Activity of B-Cell IgD Low (BDL) Regulatory B Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 General Reagents
2.3 Mice
2.4 Assessing BDL and Treg Cell Surface Phenotype by Flow Cytometry
2.5 Fluorescence Activated Cell Sorting (FACS) and Magnetic Bead Enrichment
2.6 Reagents for In Vitro Treg Proliferation Assay
2.7 Reagents for the In Vivo Treg Expansion and Maintenance Assays
3 Methods
3.1 Detecting the BDL Cell Surface Phenotype by Flow Cytometry
3.2 Detection of Treg by Flow Cytometry
3.3 FACS Purification of BDL
3.4 CD4+Foxp3EGFP Treg FACS Purification
3.5 In Vitro Treg Proliferation Assay
3.6 In Vivo Treg Expansion Assay
3.7 In Vivo Treg Maintenance Assay
3.8 Assess BDL Activity in Mouse Models of Disease
4 Notes
References
Part III: Methods for the Ex Vivo Expansion and Molecular Characterization of IL-10 Producing B Cells
Chapter 13: Use of Toll-Like Receptor (TLR) Ligation to Characterize Human Regulatory B-Cells Subsets
1 Introduction
2 Materials
2.1 General Equipment
2.2 Buffers and Media
2.3 Antibodies, Cytokines, and Other Reagents
2.4 Software
3 Methods
3.1 Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) by Density Centrifugation and Cryopreservation
3.1.1 Isolation of PBMCs Via Density Gradient Centrifugation
3.1.2 Cryopreservation of Isolated PBMCs
3.2 Thawing and Count of Cryopreserved PBMCs
3.3 Human B-Cell Isolation
3.4 Cell Resting and Cell Stimulation through TLR Ligands
3.5 Quantification of Cytokine Production Using Flow Cytometry
3.5.1 B-Cell Phenotyping and Quantification of Intracellular Cytokine Production by Flow Cytometry
3.5.2 Quantification of Cytokine Production by Cytometric Bead Array (CBA)
4 Notes
References
Chapter 14: Use of Cocultures for the Study of Cellular Interactions Influencing B-Cell Regulatory Functions
1 Introduction
2 Materials
2.1 Equipment
2.2 Mice
2.3 Buffers, Solutions and Media
2.4 Reagents and Antibodies
2.5 Other
3 Methods
3.1 Cell Preparation
3.1.1 Mouse B-Cell Purification
3.1.2 BMMC Differentiation
3.1.3 PDMC Isolation and Expansion
3.1.4 Purification of MDSCs
3.2 Cocultures
3.2.1 B/MC Coculture
3.2.2 B/MDSC Coculture
3.3 Evaluation of MC and MDSC Ability to Induce IL-10 Production in B Cells
3.3.1 Real-Time qPCR for Il-10 Gene Expression
3.3.2 ELISA for IL-10
3.3.3 Intracellular Staining (ICS) for IL-10
3.4 Contact-Dependent Mechanisms and the Role of Soluble Mediators
3.4.1 Transwell Assay
3.4.2 Conditioned Media
3.4.3 Blocking Antibodies
3.4.4 Use of Genetically Deficient Mice
4 Notes
References
Chapter 15: Use of Inhibitory Compounds to Dissect the Molecular Pathways Involved in Regulatory B-Cell Differentiation
1 Introduction
2 Materials
2.1 Mice
2.2 Magnetic-Activated Cell Sorting (MACS)
2.3 Cell Culture and Treatment
2.4 Intracellular Phospho-Staining
3 Methods
3.1 Isolation of Splenic B Cells by Negative Selection
3.1.1 Biotinylation of Magnetic Beads
3.1.2 Preparation of Anti-FITC-Streptavidin
3.1.3 Isolation of Murine Splenocytes
3.1.4 MACS Negative Selection of Splenic B Cells
3.2 Induction of IL-10 Production in Splenic B Cells by SCFAs and Use of Inhibitors
3.2.1 Induction of Bregs by pentanoate and CpG-2395
3.2.2 Determination of Optimal Working Concentration of Inhibitory Compounds
3.2.3 IL-10 Detection
3.3 Staining for Intracellular Phospho-Proteins
4 Notes
References
Chapter 16: Methods to Study the Transcriptome of Regulatory B Cells
1 Introduction
2 Materials
2.1 General Equipment
2.2 RNA Isolation
2.3 cDNA Reverse Transcription
2.4 Gene Quantification by RT-qPCR
2.5 Gene Quantification by BioMark HD System
2.6 Gene Quantification by Nanostring nCounter
2.7 Gene Quantification by Microarrays
3 Methods
3.1 RNA Isolation from B Cells and cDNA Synthesis
3.2 Fluorescence-Based Quantitative Real-Time PCR (qRT-PCR)
3.3 BioMark HD System
3.4 NanostringΒ΄s nCounter Technology
3.5 Microarrays
4 Notes
References
Chapter 17: Purification of Murine IL-10+ B Cells for Analyses of Biological Functions and Transcriptomics
1 Introduction
2 Materials
2.1 Equipment
2.2 Mice
2.3 Cell Preparation, Culture, and Stimulation
2.4 Cell Immunofluorescence Staining, Sorting, and Enrichment
2.5 RNA Profiling of B10 Cells
2.6 Ex Vivo Function of CD9+ B Cells
2.7 Reconstitution of B-Cell-Deficient Mice and Contact Hypersensitivity (CHS) Assay
3 Methods
3.1 B-Cell Preparation and Culture
3.1.1 Isolation of B Cells from the Spleen
3.1.2 Isolation of Peritoneal B Cells
3.1.3 B-Cell Stimulation for B10 and B10pro Cell Analysis
3.1.4 Differentiation of B10 cells from CD9+ B Cells
3.2 Flow Cytometry Analysis of CD9+ Breg Cells
3.3 RNA Profiling of CD9+ B10 cells for the Analysis of the Transcriptome
3.3.1 Induction and Isolation of CD9+IL-10+ B Cells
3.3.2 RNA Preparation
3.3.3 RNA Profiling
3.4 Ex Vivo Activity of CD9+ B Cells
3.4.1 T-Cell Proliferation Assay
3.4.2 Evaluation of Cell Contact Contribution on the T-Cell Proliferation Assay
3.5 In Vivo Activity of CD9+ B Cells
4 Notes
References
Chapter 18: il-10 Gene Locus DNA Methylation in Regulatory B Cells
1 Introduction
2 Materials
2.1 General Equipment
2.2 General Kits and Reagents
2.3 Reagents for Bisulfite Cloning Sequencing and Pyrosequencing
3 Methods
3.1 Preliminary Steps
3.1.1 Bisulfite Conversion of DNA and Quantification
3.1.2 Region of Interest (ROI) Identification
3.2 Cloning Bisulphite Sequencing PCR (Cloning BSP)
3.2.1 Primer Design
3.2.2 PCR of Bisulfite-Converted DNA
3.2.3 Cloning, Plasmid Extraction, and Sequencing
3.2.4 Analysis of Methylation
3.3 Pyrosequencing Bisulphite Sequencing PCR (pyrosequencing BSP)
3.3.1 Assay Design
3.3.2 PCR
3.3.3 Sequencing and Analysis
3.4 Alternative Methods for Bisulfite-Based Methylation Studies
4 Notes
References
Chapter 19: B-Cell Commitment to IL-10 Production: The VertX Il10egfp Mouse
1 Introduction
2 Materials
2.1 Mice
2.2 Equipment
2.3 Buffers and Reagents for Cell Isolation from Different Tissues
2.4 Reagents for the Identification of GFP+ B Cells by Flow Cytometry
3 Methods
3.1 Isolation of Cells from Several Tissues
3.1.1 Isolation of Spleen and Mesenteric Lymph Node Cells
3.1.2 Isolation of Intestinal Lamina propria Mononuclear Cells
3.1.3 Isolation of Peritoneal Cells
3.1.4 Isolation of Peripheral Blood Mononuclear Cells
3.2 Identification of GFP+ B Cells by Flow Cytometry
3.2.1 Staining of Surface Markers for Flow Cytometry
3.2.2 Staining Intracellular Cytokines for Flow Cytometry
3.2.3 Special Compensation Controls for Flow Cytometry
3.3 Functional Analysis of GFP+ B Cell In Vivo and In Vitro
3.3.1 GFP+ B Cells in exGF VertX Mice
3.3.2 Comparison of GFP Expression between Wild-Type and Gene-Modified B Cells
4 Notes
References
Part IV: Study of Regulatory B Cells in Pathological Settings
Chapter 20: Regulatory B Cells in Experimental Mouse Models of Arthritis
1 Introduction
2 Materials
2.1 Antigen-Induced Arthritis
2.2 Collagen-Induced Arthritis
2.3 Adoptive transfer of T2-MZPs
2.4 In Vitro Suppression Assay
3 Methods
3.1 Induction of Antigen-Induced Arthritis
3.2 Induction of Collagen-Induced Arthritis
3.3 Adoptive Transfer of T2-MZPs (In Vivo Suppression Assay)
3.3.1 Purification of T2-MZP B Cells
3.3.2 Adoptive Transfer of T2-MZP B Cells
3.4 In Vitro Suppression Assay
4 Notes
References
Chapter 21: Regulatory and IgE+ B Cells in Allergic Asthma
1 Introduction
1.1 Allergic Asthma
1.2 B Cells
1.3 IgE+ B Cells
1.3.1 IgE+ B-Cell Kinetics and Compartmental Distribution
1.4 Regulatory B Cells (Bregs)
1.4.1 Development of Bregs
1.4.2 Functions of Bregs
1.4.3 IL-10 Production by Bregs
1.4.4 Role of Bregs in Allergic Disease
1.4.5 Phenotypes of Bregs
CD1d+CD5+ Bregs
CD5+FoxP3+ Bregs
CD24+CD38+ Bregs
CD24+CD27+ Bregs
Other Bregs Phenotypes
1.5 Summary
2 Materials
2.1 Selection of Participants with Mild Allergic Asthma
2.1.1 Skin Prick Test
2.1.2 Spirometry and Methacholine Challenge Test
2.1.3 Allergen Inhalation Challenge
2.2 Sample Collection and Tissue Preparation
2.2.1 Peripheral Blood Collection and Processing
2.2.2 Bone Marrow Aspiration and Processing
2.2.3 Tonsil biopsy Processing and Cell Isolation
2.2.4 Sputum Induction and Processing
2.3 Circulating Protein Measurements
2.4 Flow Cytometry Staining
2.5 B-Cell Isolation, Culture, and Stimulation
2.5.1 B-Cell Isolation
2.5.2 B-Cell Culture and Stimulation
3 Methods
3.1 Selection of Participants with Mild Allergic Asthma
3.1.1 Determination of Allergic Status Using Skin Prick Testing
3.1.2 Measurement of lung Function Using Spirometry
3.1.3 Airway Hyperresponsiveness Testing Using the Methacholine Challenge Test
3.1.4 Inducing Symptomatic Allergic asthma Using the Inhaled Allergen Challenge
3.2 Sample Collection and Tissue Preparation
3.2.1 Collection of Venous Blood through Venipuncture
3.2.2 Aspiration of Bone Marrow Sample from the Iliac Crest
3.2.3 Homogenization of Tonsil biopsy and Cell Isolation
3.2.4 Induction of Sputum Samples
3.3 Circulating IgE and BAFF Measurements
3.4 Flow Cytometry Experiments
3.4.1 General Cell Staining Protocol for Flow Cytometry
3.4.2 Gating Strategy to Identify the Rare Population of IgE+ B Cells
3.4.3 Gating Strategy to Identify Different Regulatory B-Cell Subsets
3.5 Bregs Functional Assay
3.5.1 Isolation of Peripheral Blood CD19+ B Cells by Magnetic-Based Cell Separation
3.5.2 B-Cell Stimulation with IL-4 to Determine Functional Expression of IL-10 and FoxP3
3.6 Next Generation Methods
3.6.1 Mass Cytometry (CyTOF)
3.6.2 Multiplexed Ion Beam Imaging Time of Flight (MIBI-TOF)
4 Notes
References
Chapter 22: Regulatory B Cells in Type 1 Diabetes
1 Introduction
2 Materials
2.1 Mice
2.2 Equipment
2.3 Reagents for Tissue Preparation
2.4 Reagents for Cell Culture and Flow cytometry Analysis
3 Methods
3.1 Tissue Preparation
3.1.1 Isolation of Cells from the Spleen
3.1.2 Isolation of Cells from the Bone Marrow
3.1.3 Isolation of Cells from Pancreatic Lymph Nodes
3.1.4 Isolation of Cells from WT NOD Pancreatic Islets
3.2 Phenotypical Characterization of Regulatory B Cells
3.2.1 Cell Stimulation
3.2.2 Cell Staining for the Detection of Surface Markers on B Cells
3.2.3 Cell Staining for the Detection of IL-10-Expressing B Cells
3.3 Functional Characterization of Regulatory B Cells
3.3.1 B-Cell Regulation of BM-DCs Function
Set up of the B Cell-DC Coculture
Evaluation of DC Phenotype Following B Cell-DC Coculture
Evaluation of DC Functionality Following B Cell-DC Coculture
3.3.2 B-Cell Regulation of Antigen-Specific CD8+ T-Cell Function: B Cell-DC-CD8+ T-Cell Cocultures
4 Notes
References
Chapter 23: Analysis of Regulatory B Cells in Experimental Autoimmune Uveitis
1 Introduction
2 Materials
2.1 General Instrumentation
2.2 Tissue Dissection and Cell Culture Reagents
2.3 Reagents for the Study of i35-Breg Cells
2.4 Mice and Immunization Reagents
2.5 Reagents for the Assessment of Uveitis
3 Methods
3.1 Induction of EAU
3.2 Assessment of Uveitis and Pathological Score
3.3 Isolation of Regulatory or Inflammatory Cells from the Neuroretina
3.4 Isolation of Splenocytes
3.5 Regulatory B-Cell Enrichment and Isolation
3.5.1 Induction and Isolation of CD138+ Cells
3.5.2 Detection of IL-35-Producing CD138+ Cells by Intracellular Cytokine Staining
3.6 Adoptive transfer of IL-35-Producing B Cells in Recipient Uveitis Mice
4 Notes
References
Chapter 24: Purification and Immunophenotypic Characterization of Human CD24hiCD38hi and CD24hiCD27+ Regulatory B Cells in Tra...
1 Introduction
2 Materials
2.1 General Equipment
2.2 Buffers and Reagents
2.3 Antibodies and Reagents for Flow Cytometry
2.4 Reagents for Cell Enrichment and Isolation
2.5 Reagents for Cell Culture and Assay
2.6 Reagents for Serum Anti-HLA Antibodies Detection
2.7 Enzyme-Linked Immunosorbent Spot (ELISPOT) Assay for the Detection of Anti-HLA Cellular Responses by IFN-Ξ³
3 Methods
3.1 Peripheral Blood Mononuclear Cells (PBMCs) Isolation
3.2 B-Cell Subsets Phenotyping
3.3 Isolation of B-Cell Subsets by FACS Sorting
3.4 CD4+CD25- T-Cell Enrichment
3.4.1 CD4+ T-Cell Enrichment
3.4.2 CD4+CD25- T-Cell Enrichment
3.5 B-Cell Functional Assays
3.5.1 Suppression Assay: T-Cell Proliferation
3.5.2 Suppression Assay: Cytokines Quantification by Intracellular Cytokine Staining
3.6 HLA-Related Testing
3.6.1 Detection of Serum Anti-HLA Antibodies
3.6.2 Assessment of Anti-HLA Cellular Response
4 Notes
References
Index