BASOPHILS AND MAST CELLS : methods and protocols.

Acknowledgements
Preface to the Second Edition
Preface to the First Edition
Contents
Contributors
Part I: Introduction and Historical Background
	Chapter 1: Paradigm Shifts in Mast Cell and Basophil Biology and Function: An Emerging View of Immune Regulation in Health and...
		1 Introduction
		2 Novel Roles for Mast Cells and Basophils: Protecting the  Host
			2.1 An Unexpected Protective Role of Mast Cells in Atopic Dermatitis
			2.2 Protection by Basophils to Reinfection (Hookworms, Ticks)
			2.3 The Protective Effect of Mast Cell and Basophil Proteases
		3 Unexpected Physiopathological Roles of Mast Cells and Basophils
			3.1 Adverse Effects of Mast Cell Protection in Atopic Dermatitis: The Atopic March
			3.2 An Unexpected Role for Mast Cells in Obesity
			3.3 Mast Cells in Atherosclerosis
			3.4 Mast Cells in Tumor Growth and Development
			3.5 Mast Cells and Basophils in Autoimmune Diseases
		4 The Immunomodulatory Role of Mast Cells and Basophils
			4.1 Recent Advances in Mast Cell Immune Modulation
			4.2 Basophils and B Cells: Boosting the Humoral Response
		5 Concluding Remarks
		References
Part II: Obtaining the Cells: Purification, Culture, and Counting
	Chapter 2: Purification of Basophils from Peripheral Human Blood
		1 Introduction
			1.1 A Brief History of Basophil Purification Protocols
			1.2 The 1960s and 1970s: First Attempts at Enriching Basophils
			1.3 The 1980s: The 50% Purity Barrier Is Overcome, but Recoveries Remain  Low
			1.4 The 1990s: Immunomagnetic Selection Makes First Breakthroughs Possible
			1.5 The 2000s: New Emphasis on Rapid or Simplified Protocols
			1.6 Concluding Remarks
		2 Materials
			2.1 Reagents
			2.2 Supplies
			2.3 Equipment
		3 Methods
			3.1 Isolation of Basophils from Peripheral Blood
			3.2 Preparation of Cytospins
		4 Notes
		References
	Chapter 3: Mast Cell Purification Protocols
		1 Introduction
		2 Materials
			2.1 Buffers and Culture Media
			2.2 Mast Cell Staining
			2.3 Magnetic Cell Sorting
		3 Methods
			3.1 Conjugation of Dynabeads
			3.2 Tissue Dispersal and Digestion
			3.3 Mast Cell Purification
		4 Notes
		References
	Chapter 4: Generation of a Human Allergic Mast Cell Phenotype from CD133+ Stem Cells
		1 Introduction
		2 Materials
			2.1 Isolation and Culture of CD133+ Human Stem Cells
			2.2 Reagents for Determining Mast Cell Purity and Reactivity
				2.2.1 May-Grünwald-Giemsa Staining
				2.2.2 Detection of Density of FcεRI, Mast Cell Reactivity, and Kit Surface Receptor Expression by Flow Cytometry
		3 Methods
			3.1 Purification of CD133+ Stem Cells from Peripheral Blood
			3.2 Expansion of Stem Cells and Maturation of Mast Cells
			3.3 Mast Cell Staining with May-Grüwald-Giemsa by Microscopy
			3.4 Mast Cell Reactivity and Phenotyping by Flow Cytometry
		4 Notes
		References
	Chapter 5: Generation and Culture of Peripheral CD34+ Stem Cell-Derived Mast Cells (PSCMCs)
		1 Introduction
		2 Material
			2.1 Isolation and Expansion of CD34+ Cells
			2.2 Purification, Differentiation, and Culture of MCs
		3 Methods
			3.1 Isolation of CD34+ Cells with CD34 Positive Selection Kit II (Day 0)
			3.2 Expansion and Differentiation of Mast Cells (Day 0-35)
			3.3 Purification of Mast Cells with the CD117 MicroBead Kit (Day 35)
			3.4 Culture of Mast Cells (from Day 35)
		4 Notes
		References
	Chapter 6: Modulating the Human Basophil Phenotype During Its Development and Maturation: Basophils Derived from In Vitro Cult...
		1 Introduction
			1.1 Omalizumab and the Maturing Basophil
			1.2 Expression of SYK in Basophils and Other Leukocytes
			1.3 Models of Basophil Maturation
			1.4 Culture Differences
		2 Materials
			2.1 Supplies
			2.2 Buffers
			2.3 Equipment
		3 Methods
			3.1 Using Commercially Prepared CD34+ Progenitors
			3.2 Using CD34+ Cells Obtained from Leukapheresis Packs
				3.2.1 Percoll of Leukapheresis Cells (Processed Blood Enriched with Leukocytes)
				3.2.2 Positive Selection of CD34 Cells
				3.2.3 Culture
		4 Notes
		References
	Chapter 7: Generation of Mast Cells from Murine Stem Cell Progenitors
		1 Introduction
		2 Materials
			2.1 Isolation of Mouse Bone Marrow Progenitors
			2.2 Determination of BMMC Purity
				2.2.1 Toluidine Staining
				2.2.2 Flow Cytometry
		3 Methods
			3.1 Bone Marrow Cell Isolation and Mast Cell Culture
				3.1.1 Isolation and Culture of Bone Marrow Progenitors
				3.1.2 Expansion of Bone Marrow Cells and Maturation of BMMCs
			3.2 Determination of BMMC Purity
				3.2.1 Toluidine Blue Staining of Cytospin Preparations
				3.2.2 Surface Expression of FcεRI and CD117 (Kit) by Flow Cytometry
		4 Notes
		References
	Chapter 8: Integration of the Human Dermal Mast Cell into the Organotypic Co-culture Skin Model
		1 Introduction
		2 Materials
			2.1 Cell Isolation
			2.2 Purification of Dermal Mast Cells
			2.3 Cell Culture
			2.4 Organotypic Co-culture
			2.5 Preparation of Frozen Sections
			2.6 Immunohistochemistry
		3 Methods
			3.1 Mast Cell Isolation, Purification, and Culture
			3.2 Fibroblast Isolation and Culture
			3.3 Keratinocyte Isolation and Culture
			3.4 Skin Equivalent (SE)
			3.5 Airlifting
			3.6 Preparation of Frozen Sections
			3.7 Immunohistochemistry
		4 Notes
		References
	Chapter 9: The Absolute Basophil Count
		1 Introduction
		2 Materials
			2.1 Manual Basophil Counting
				2.1.1 Hemocytometer Method
				2.1.2 Peripheral Blood Smears
			2.2 Automated Basophil Counting Using Flow Cytometry
		3 Methods
			3.1 Manual Absolute Basophil Counts in Whole Blood Stained with Kimura Stain Using Cell-Counting Chambers
			3.2 Manual Absolute Basophil Counting Procedure Using a Peripheral Blood Smear
				3.2.1 Prepare a Peripheral Blood Smear
				3.2.2 Staining a Peripheral Blood Smear with May-Grünwald-Giemsa Stain
				3.2.3 Staining a Peripheral Blood Smear with Wright Stain
				3.2.4 Evaluation of Stained Peripheral Blood Smears
			3.3 Absolute Basophil Counts in Whole Blood Using Flow Cytometry Labeling
				3.3.1 Double-Platform Counting Method
				3.3.2 Single-Platform Microbead-Based Method
				3.3.3 Single-Platform Volumetric Method
		4 Notes
		References
Part III: Basophil/Mast Cell Lines
	Chapter 10: Mast Cell and Basophil Cell Lines: A Compendium
		1 Mast Cells and Basophils, an Introduction
			1.1 Human Mast Cell Lines
			1.2 HMC-1 and HMC-1 Sublines
			1.3 LAD Cell Lines
			1.4 LUVA Cell Line
			1.5 ROSAKIT WT Cell Line, ROSAKIT D816V, and ROSAKIT D816V-Gluc Subclones
			1.6 KU812
		2 Rodent Cell Lines
			2.1 RBL Cell Lines
			2.2 NCL-2 Cell Line
		3 Concluding Remarks
		References
	Chapter 11: Use of Humanized RBL Reporter Systems for the Detection of Allergen-Specific IgE Sensitization in Human Serum
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Supplies
			2.3 Equipment
		3 Method
			3.1 General Cell Culture
			3.2 (Optional) Treatment of Serum Before Sensitization
			3.3 Sensitization
			3.4 Stimulation
			3.5 Measurement of Activation
				3.5.1 Fluorescence Assay with NFAT-DsRed
				3.5.2 Luciferase Assay (EXiLE Test, See Note 9)
		4 Notes
		References
	Chapter 12: Use of Humanized Fluorescent Reporter Cell Line RBL NFAT-DsRed for the Detection of Allergen-Specific IgE in Patie...
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Supplies
			2.3 Equipment
		3 Methods
			3.1 General Cell Culture
			3.2 Treatment of Serum Before Sensitization
			3.3 Sensitization
			3.4 Protein Blocking
			3.5 Stimulation
			3.6 Measurement of Activation
			3.7 Data Analysis
		4 Notes
		References
	Chapter 13: NPY-mRFP Rat Basophilic Leukemia (RBL) Reporter: A Novel, Fast Reporter of Basophil/Mast Cell Degranulation
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Supplies
			2.3 Equipment
		3 Method
			3.1 General Cell Culture
			3.2 [Optional] Treatment of Serum Before Sensitization
			3.3 Sensitization
			3.4 Stimulation
			3.5 Fluorescence Assay with NPY-mRFP RBL
		4 Notes
		References
	Chapter 14: Use of Engineered Nanoparticles (ENPs) for the Study of High-Affinity IgE FcεRI Receptor Engagement and Rat Basoph...
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Supplies
			2.3 Equipment
		3 Methods
			3.1 General Cell Culture
			3.2 β-Hexosaminidase Assay
				3.2.1 Sensitization
				3.2.2 Stimulation
				3.2.3 Measurement of Activation
			3.3 Polystyrene Nanoparticle Preparation
				3.3.1 Polystyrene Surface Modified with DNP-BSA
				3.3.2 Polystyrene Surface Modified with DNP-BSA and IgE
		4 Notes
		References
Part IV: Diagnostic Applications
	Chapter 15: Flow Cytometric Allergy Diagnosis: Basophil Activation Techniques
		1 Introduction
		2 Materials
			2.1 Blood Sample
			2.2 Reagents
		3 Methods
			3.1 Flow Cytometric Basophil Activation Technique: Membrane Markers (Method 1)
				3.1.1 Alexa Fluor 405 Labeling of Mouse Monoclonal Anti-human IgE
				3.1.2 Preparation of Positive and Negative Controls (Exclusion of Nonresponders)
				3.1.3 Allergen Procedure
			3.2 HistaFlow: Basophil Activation Technique with Intracellular Staining of Histamine (Method 2)
				3.2.1 Preparation of Positive and Negative Controls (Exclusion of Nonresponders)
				3.2.2 Allergen Procedure
			3.3 Compensation Procedure
			3.4 Calibration Procedure
		4 Notes
		References
	Chapter 16: Basophil Activation Experiments in Immediate Drug Hypersensitivity: More Than a Diagnostic Aid
		1 Introduction
		2 Technical Principles
		3 Clinical Applications
		4 Exploring Mechanistic Endotypes
		5 Detection and Identification of Antibody Recognition Structures
		6 Drug Challenge Tests (DCTs): From Bed to Bench?
		7 Excipients
		8 Desensitization
		9 Conclusion
		References
	Chapter 17: Basophil Activation Techniques: Staining of Exteriorized Granule Matrix
		1 Introduction
		2 Materials
			2.1 Blood Sample
			2.2 Reagents
		3 Methods
			3.1 Labeling Procedure
			3.2 Control of Available Basophils and Their Stimulation (Exclusion of Nonresponders)
			3.3 Allergen Procedure
		4 Notes
		References
	Chapter 18: Measurement and Functional Analysis of the Mas-Related G Protein-Coupled Receptor MRGPRX2 on Human Mast Cells and ...
		1 Introduction
		2 Materials
			2.1 Mast Cells
			2.2 Blood for Basophil Activation Test
			2.3 Reagents
		3 Methods
			3.1 Expression of MRGPRX2 on Cultured Mast Cells (PBCMCs)
			3.2 Mast Cell Activation via MRGPRX2
			3.3 MRGPRX2 on Basophils After the So-Called Conditioning
		4 Notes
		References
	Chapter 19: Mast Cell Activation Test (MAT)
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Human Biological Material
			2.3 Reagents
				2.3.1 Human Peripheral Blood-Derived Mast Cell Differentiation
				2.3.2 Degranulation Assay and Flow Cytometry Staining
				2.3.3 Disinfectant
			2.4 Solution and Media
			2.5 Software
		3 Methods
			3.1 Human Peripheral Blood-Derived Mast Cell Differentiation (8-10 Weeks)
				3.1.1 Peripheral Blood Mononuclear Cell (PBMC) Isolation from Peripheral Blood
				3.1.2 CD117+ Cell Isolation from PBMCs
				3.1.3 Cell Culture
			3.2 Degranulation (48 h) (Fig. 2)
				3.2.1 Day 1: Mast Cell Overnight Sensitization with Sera (1:10) or IgE (1 μg/mL) in a 96-Well Plate
				3.2.2 Day 2: Activation with Allergen or Anti-IgE
				3.2.3 Flow Staining
				3.2.4 Flow Analysis Data Using FlowJo Software (Fig. 3)
		4 Notes
		References
Part V: Whole Cells: Functional Studies
	Chapter 20: Quantity and Quality of Basophil RNA Depend on the RNA Extraction Technique
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Samples
			3.2 RNA Purification
		4 Notes
		References
	Chapter 21: Measuring Histamine and Cytokine Release from Basophils and Mast Cells
		1 Introduction
			1.1 Histamine
			1.2 Cytokines
		2 Materials
			2.1 Histamine Release and Quantification
			2.2 Detection of Intracellular Histamine by Flow Cytometry
			2.3 Cytokine Release
		3 Methods
			3.1 Histamine Release and Quantification
				3.1.1 Histamine Release in Whole Blood
				3.1.2 Histamine Release from Purified Basophils
				3.1.3 Histamine Release from In Vitro Cultured Human Mast Cells
				3.1.4 Histamine Release from In Situ Mast Cells
				3.1.5 Histamine Quantification
			3.2 Detection of Intracellular Histamine in LAD2 Cells Using Flow Cytometry
				3.2.1 DAO-PE Preparation
				3.2.2 Cell Activation
				3.2.3 Intracellular Staining
				3.2.4 Flow Cytometry Analysis
			3.3 Cytokine Release and Detection
				3.3.1 Cytokine Release with Purified Basophils or In Vitro Cultured Human Mast Cells
				3.3.2 Cytokine Release from Skin Mast Cells
				3.3.3 Cytokine Detection
		4 Notes
		References
	Chapter 22: Detection of Mast Cells and Basophils by Immunohistochemistry
		1 Introduction
		2 Materials
			2.1 Antibodies
			2.2 Fixatives, Tissue Processing, and Slide Preparation
			2.3 Reagents for Antigen Retrieval and Immunohistochemistry
			2.4 Equipment
		3 Methods
			3.1 Fixation and Processing of Tissues
			3.2 Preparation and Fixation of Cytocentrifuge Preparations
			3.3 De-waxing and Rehydration of Tissue Sections
			3.4 Heat-Induced Epitope Retrieval
			3.5 Immunohistochemistry with a Single Primary Antibody
			3.6 Double Labeling Immunohistochemistry for MCT and MCTC Subpopulations
		4 Notes
		References
	Chapter 23: Detection of Basophils and Other Granulocytes in Induced Sputum by Flow Cytometry
		1 Introduction
		2 Materials
			2.1 Sputum Induction
			2.2 Sputum Processing
			2.3 Cell Counting by Flow Cytometry
			2.4 Surface Staining for Flow Cytometry
		3 Methods
			3.1 General Information and Preparation for Sputum Induction
			3.2 Sputum Induction
			3.3 Sputum Processing
			3.4 Cell Counting by Flow Cytometry
			3.5 Surface Staining for Flow Cytometry
			3.6 Gating Strategy for Basophils, Eosinophils, and Neutrophils in Flow Jo or Equivalent
		4 Notes
		References
	Chapter 24: Mast Cell Migration and Chemotaxis Assayed by Microscopy
		1 Introduction
		2 Materials
			2.1 Chemicals and Kits
			2.2 Buffers and Media
			2.3 Mast Cells
			2.4 Other Materials
			2.5 Equipment
			2.6 Software
		3 Methods
			3.1 Motility Assay
			3.2 Mast Cell Chemotaxis in μ-Slide Chemotaxis3D Chambers
			3.3 Chemotaxis in Homemade Chambers
			3.4 Tracking Cells
			3.5 Presenting Chemotaxis Data
		4 Notes
		References
	Chapter 25: Basophil Stimulation and Signaling Pathways
		1 Introduction
		2 Materials
			2.1 Chemicals and Kits
			2.2 Buffers for Cell Culture and Stains
			2.3 Basophils
			2.4 Equipment and Other Materials
		3 Methods
			3.1 Basophil Stimulation Assay: General Procedure
			3.2 Histamine Release Analysis
			3.3 Eicosanoid and Cytokine Release Analysis
			3.4 Measurement of Cytosolic Free Calcium Concentrations by Spectrofluorophotometer or Flow Cytometer
			3.5 Determination of Activation Pathways by Flow Cytometry
			3.6 Signal Transduction Experiments and Preparation of Samples for SDS-PAGE and Western Blotting
		4 Notes
		References
	Chapter 26: Gene Silencing Approaches in Mast Cells and Primary Human Basophils
		1 Introduction
		2 Materials
			2.1 Cells
			2.2 Reagents
		3 Methods
			3.1 Transfection
			3.2 Analysis of Gene Silencing
		4 Notes
		References
	Chapter 27: Identification and Immunophenotypic Characterization of Normal and Pathological Mast Cells
		1 Introduction
		2 Materials
		3 Methods
			3.1 Cell Suspension Preparation
				3.1.1 Specific Procedures for Bone Marrow Samples
				3.1.2 Specific Procedures for High Diluted Samples (Organic Fluids)
				3.1.3 Other Samples
			3.2 Antigen Staining
				3.2.1 Staining Procedure for Surface Antigens Only
				3.2.2 Combined Staining Protocol for Intracellular and Surface Membrane Antigens
			3.3 Instrument Setup and Data Acquisition
			3.4 Data Analysis
				3.4.1 Mast Cell Identification and Enumeration
				3.4.2 Mast Cell Phenotype Assessment
			3.5 Expected Results
				3.5.1 Mast Cell-Committed Precursors
				3.5.2 Mature Resting BMMC Phenotype Under Normal and Reactive Conditions
				3.5.3 Activated Immunophenotype of Normal MCs
				3.5.4 MC Immunophenotype in Systemic Mastocytosis
		4 Notes
		References
Part VI: Whole Organism: Disease and Knockout Models
	Chapter 28: Mast Cell-Mediated Reactions In Vivo
		1 Introduction
		2 Materials
			2.1 PCA
			2.2 PSA
			2.3 CHS Materials
		3 Methods
			3.1 PCA
				3.1.1 Day 0 (Passive Sensitization)
				3.1.2 Day 1 (Cutaneous Anaphylaxis Elicitation)
				3.1.3 Assessment of Ear Swelling
				3.1.4 Quantifying Evans Blue Dye Extravasation
			3.2 PSA
				3.2.1 Day 0 (Passive Sensitization)
				3.2.2 Day 1 (Systemic Anaphylaxis Elicitation)
				3.2.3 Body Temperature Measurement
				3.2.4 Assessment of Mast Cell Mediator Release
			3.3 CHS (See Note 12)
				3.3.1 Day 5 (Sensitization)
				3.3.2 Day 0 (Challenge)
		4 Notes
		References
	Chapter 29: Identification of Murine Basophils by Flow Cytometry and Histology
		1 Introduction
		2 Materials
			2.1 Flow Cytometry Components
			2.2 Histology Components
		3 Methods
			3.1 Flow Cytometry
			3.2 Histology
		4 Notes
		References
Index