Eosinophils: Methods and Protocols

Description

Preface Contents Contributors Chapter 1: Molecular Biology of Eosinophils: Introduction 1 Introduction 2 Accumulation and Fate 3 Secretion and Receptors 4 Clinical Overview 5 Functional Studies in Animal Models 6 Role in Innate and Adaptive Immunity 7 Conclusions References Chapter 2: Functionally Active Eosinophil Purification from Peripheral Blood 1 Introduction 2 Materials 2.1 Eosinophil Isolation 2.2 Assessing Population Purity via Cytospin Staining 2.3 Evaluating Population Purity and Cell Activation State via Flow Cytometry 3 Methods 3.1 Eosinophil Isolation 3.2 Identification of Pure Populations via Cytospin Differential 3.3 Evaluating Population Purity and Cell Activation State via Flow Cytometry 4 Notes References Chapter 3: Eosinophil Purification from Peripheral Blood of Rhesus Monkeys 1 Introduction 2 Materials 2.1 Buffer and Solutions 2.2 Reagents 2.3 Equipment 3 Methods 3.1 Rhesus Macaque Blood Draw 3.2 Eosinophil Purification 3.3 Eosinophil Viability Measurement by Flow Cytometry 4 Notes References Chapter 4: Generation of Mouse Eosinophils in Tissue Culture from Unselected Bone Marrow Progenitors 1 Introduction 2 Materials 2.1 Isolation of Unselected Progenitor Cells from Mouse Bone Marrow 2.2 Differentiation and Culture of Eosinophils from Bone Marrow Progenitor Cells 2.2.1 Bone Marrow-Derived Eosinophil (bmEos) Base Media (500 mL Total Volume) 2.2.2 Concentrated Cytokine Stocks Diluted in Base Medium 2.3 Detection and Enumeration of bmEos by Cytochemical Staining 2.4 Detection and Enumeration of bmEos by Flow Cytometry 2.5 Equipment 3 Methods 3.1 Preparation of Base Media and Concentrated Cytokine Stocks 3.2 Isolation of Unselected Progenitor Cells from Mouse Bone Marrow 3.3 Differentiation and Culture of Eosinophils from Bone Marrow Progenitor Cells 3.4 Detection and Enumeration of bmEos by Cytochemical Staining 3.5 Detection and Enumeration of bmEos by Flow Cytometry 4 Notes References Chapter 5: Detection of Mouse Eosinophils in Tissue by Flow Cytometry and Isolation by Fluorescence-Activated Cell Sorting (FA... 1 Introduction 2 Materials 2.1 Isolation of Lungs from Allergen-Challenged or IL5tg Mice 2.2 Preparation of Single-Cell Suspensions 2.3 Antibodies for Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS) 2.4 Equipment/Computer Resources 3 Methods 3.1 Isolation of Lungs from Allergen-Challenged or IL5tg Mouse 3.2 Preparation of Single-Cell Suspensions 3.3 Flow Cytometric Analysis 3.4 Fluorescence-Activated Cell Sorting (FACS) 4 Notes References Chapter 6: Mutant Mice and Animal Models of Airway Allergic Disease 1 Introduction 1.1 Mouse Strains to Study the Role of Eosinophils in Allergic Responses 1.1.1 The PHIL Mouse 1.1.2 The dblGATA Mouse 1.1.3 The eos-CRE Mouse 1.1.4 The Cd34-/- Mouse 1.1.5 The Rorasg/sg Mouse 1.2 Mouse Models to Study the Role of Eosinophils in Disease 1.2.1 Ovalbumin Model of Asthma 1.2.2 House Dust Mite Model of Asthma 1.2.3 Papain Model of Eosinophilia 1.2.4 Archaebacteria Antigen-Induced Allergic Airway Inflammation: A Model of Mild Eosinophilia 1.2.5 Hypersensitivity Pneumonitis as a Negative Control to Study Eosinophil Function 2 Materials 2.1 Ovalbumin Model of Asthma 2.2 House Dust Mite Model of Asthma 2.3 Papain Model of Eosinophilia 2.4 Methanosphaera stadtmanae Mixed Response of Airway Inflammation 2.5 Hypersensitivity Pneumonitis: Negative Control for Eosinophilic Responses 2.6 Assessment of AIRWAY Inflammation 2.7 Lung Inflation and Fixation for Histology 3 Methods 3.1 Ovalbumin Model Disease Induction 3.2 HDM Extract Acute and Chronic Model Disease Induction 3.3 Papain Model Disease Induction 3.4 MSS Model Disease Induction 3.5 HP Model Disease Induction 3.6 Analysis of Disease Severity: Bronchoalveolar Lavage (BAL) 3.7 Analysis of BAL Fluid: Enumeration and Differential Counts of BAL Leukocytes 3.8 Histology and Tissue Inflammation Analysis 4 Notes References Chapter 7: Eosinophils in Asthma Models to House Dust Mite for Drug Development 1 Introduction 2 Materials 3 Methods 3.1 Short Design Model (8 Days): Perform HDM Sensitization with Adjuvant 3.2 Short Design Model (8 Days): HDM Challenge 3.3 Chronic Asthma Model (31 Days): Perform HDM Sensitization 3.4 Chronic Asthma Model (31 Days): HDM Asthma Maturation 3.5 Chronic Asthma Model (31 Days): HDM Exacerbation (Fig. 1c) 3.6 Bronchoalveolar Lavage Procedure 4 Notes References Chapter 8: Analysis of Mouse Eosinophil Migration and Killing of Tumor Cells 1 Introduction 2 Materials 2.1 Isolation of Eosinophils Using Dynabeads 2.2 Concentrated Conditioned Media of Tumor Cells 2.3 Chemotaxis of Eosinophils 2.4 Cytotoxicity Assays 3 Methods 3.1 Negative Isolation of Eosinophils from Il5 Transgenic (Il5Tg) Mice 3.2 Preparation of Tumor Cell-Conditioned Media 3.3 Chemotaxis of Eosinophils Toward Tumor-Secreted Factors 3.4 Cytotoxicity of Eosinophils Toward Tumor Cells 4 Notes References Chapter 9: Murine Models of Eosinophil Function in Fungal and Viral Infections 1 Introduction 2 Materials 2.1 Development and Characterization of a Model of Asthma and Influenza That Protects the Host from Severe Influenza 2.2 Determination of the In Vivo Relevance of Eosinophils During Influenza Pathogenesis 2.3 Dynamics of Eosinophil Responses to IAV 3 Methods 3.1 Development and Characterization of a Model of Asthma and Influenza That Protects the Host from Severe Influenza 3.2 Determination of the In Vivo Relevance of Eosinophils During Influenza Pathogenesis 3.3 Dynamics of Eosinophil Responses to IAV 4 Notes References Chapter 10: Assays of Eosinophil Apoptosis and Phagocytic Uptake 1 Introduction 2 Materials 2.1 Culture of Human Eosinophils In Vitro 2.2 Cytocentrifuge Preparations of Eosinophils for Light Microscopy 2.3 Preparation for Electron Microscopy 2.4 Annexin V/Propidium Iodide Staining 2.5 Alterations in Mitochondrial Permeability 2.6 Western Blotting for Intracellular Proteins That Regulate Apoptosis 2.7 Fluorimetric Caspase Kit 2.8 Caspase Profiling Plate 2.9 Gel Electrophoresis for DNA Fragmentation 2.10 Propidium Iodide Staining for Hyplodiploid Nuclei 2.11 TUNEL Staining 2.12 Flow Cytometry-Based Phagocytosis Assay 3 Methods 3.1 Assessing Morphological Changes of Apoptosis Using Light Microscopy 3.2 Analysis of Eosinophil Morphology by Electron Microscopy 3.3 Annexin V/Propidium Iodide Staining and Flow Cytometric Analysis of Apoptosis 3.4 Measuring Mitochondrial Membrane Potential Using MitoCapture 3.5 Western Blotting for Caspases and Apoptotic Proteins 3.6 Fluorometric Homogeneous Caspase Assay 3.7 Caspase Profiling Assay 3.8 Assessing Nuclear Changes During Apoptosis: Gel Electrophoresis of DNA 3.9 Hypodiploid DNA Content 3.10 TUNEL Staining for DNA Breaks 3.11 Assessing Phagocytic Uptake of Apoptotic Cells: Culture of Monocyte-Derived Macrophages 3.12 Flow Cytometry-Based Phagocytosis Assay 4 Notes References Chapter 11: In Vivo Eosinophil Expansion Using Gene Transfer by Electroporation 1 Introduction 2 Materials 3 Methods 3.1 Prepare IL-5 Plasmid DNA 3.2 IL-5 Plasmid DNA Transfer into Muscle by Electroporation In Vivo 3.3 Flow Cytometric Analysis of Peripheral Blood Cells 4 Notes References Chapter 12: Observation and Quantification of Eosinophil Motility 1 Introduction 2 Materials 2.1 DIC Video Microscopy 2.2 Bead-Clearing Motility Assay 3 Methods 3.1 Differential Interference Contrast Video Microscopy 3.2 Bead-Clearing Motility Assay 4 Notes References Chapter 13: Isolation and Functional Aspects of Eosinophil-Derived Exosomes 1 Introduction 2 Materials 2.1 Eosinophil Purification from Human Peripheral Blood 2.2 Eosinophil Culture 2.3 Exosome Isolation 2.4 Functional Assays 3 Methods 3.1 Eosinophil Isolation 3.2 Eosinophil Culture 3.3 Exosome Purification 3.4 Exosome Analyses 3.4.1 Exosome Size Determination and Quantification 3.4.2 Eosinophil-Derived Exosome Staining 3.4.3 Suspension Cell Functional Assays 3.4.4 Adherent Cell Functional Assays 4 Notes References Chapter 14: Interactions of Eosinophils with Nerves 1 Introduction 2 Materials 2.1 General Methods 2.2 Culture Plates 2.3 Optimizing Neuronal Adherence 2.4 Preparation of Serum-Free Media 2.5 Growth Factors and Additives 2.6 Preparation of Parasympathetic Ganglia from Guinea Pigs, Rats, and Mice 2.7 Coculture of Parasympathetic Nerves with Eosinophils and RNA Harvest FROM Parasympathetic Ganglia 2.8 Preparation of Mouse, Guinea Pig, or Rat Dorsal Root Ganglia (DRG) for Culture 2.9 Preparation of Mouse, Guinea Pig, or Rat Vagal Ganglia for Culture 2.10 Retrograde Labeling to Identify and Isolate Pulmonary-Specific Afferent Nerves in Vagal Ganglia 2.11 Guinea Pig Eosinophil isolation (for Human Eosinophils See Chapter 2) 2.12 Mouse Eosinophil Isolation 2.13 Coculture of Sympathetic Nerves with Eosinophils and Quantification of Nerve Length and Branching 3 Methods 3.1 Preparation of Parasympathetic Ganglia from Guinea Pigs 3.2 Preparation of Parasympathetic Ganglia from Human Trachea 3.3 Coculture of Parasympathetic Nerves with Eosinophils and RNA Harvested from Parasympathetic Ganglia 3.4 Preparation of Mouse, Guinea Pig, or Rat Dorsal Root Ganglia (DRG) for Culture 3.5 Preparation of Mouse, Guinea Pig, or Rat Vagal Ganglia for Culture 3.6 Retrograde Labeling to Identify and Isolate Pulmonary-Specific Afferent Nerves in Vagal Ganglia 3.7 Guinea Pig Eosinophil Isolation (for Human Eosinophils See Chapter 2) 3.8 Mouse Eosinophil Isolation 3.9 Coculture of Sympathetic Nerves with Eosinophils and Quantification of Nerve Length and Branching 4 Notes References Chapter 15: Measurement of Eosinophil Kinetics In Vivo 1 Introduction 2 Materials 2.1 Eosinophil Separation 2.2 Eosinophil Imaging Using SPECT-CT 3 Methods 3.1 Eosinophil Isolation and Imaging 3.2 Image Analysis for Quantifying Eosinophil Uptake 4 Notes References Chapter 16: Detection of Eosinophil Extracellular DNA Traps 1 Introduction 2 Materials 2.1 Semiquantitative Fluorimetric Assay 2.2 Qualitative Fluorescent Microscopy Assay 3 Methods 3.1 Semiquantitative Fluorimetric Analysis 3.2 Qualitative Fluorescent Microscopy Analysis 4 Notes References Chapter 17: Eosinophil Shape Change and Secretion 1 Introduction 2 Materials 2.1 Actin Staining in Adherent Eosinophils 2.2 In Vitro Degranulation Assay 2.3 Ex Vivo Degranulation Assay 2.4 o-Phenylenediamine (OPD) EPX Enzymatic Activity Assay 2.5 EPX Enzyme-Linked Immunosorbent Assay (ELISA) 3 Methods 3.1 Actin and Nuclear Staining in Adherent Eosinophils to Detect Eosinophil Shape Change 3.2 In Vitro Degranulation Assay 3.3 Ex Vivo Degranulation Assay 3.4 OPD Assay for EPX Activity 3.5 EPX ELISA 4 Notes References Chapter 18: Update on Eosinophil Interaction with Mast Cells: The Allergic Effector Unit 1 Introduction 2 Materials 2.1 Culture Medium for BMMCs and BMEos 2.2 Cytokines Required for Culturing BMMCs 2.3 Cytokines Required for Culturing BMEos 2.4 Buffers and Solutions 2.5 Other Requirements 3 Methods 3.1 Ex Vivo Differentiation and Culture of MC and Eos from Adult Mouse Bone Marrow 3.1.1 Harvesting and Culture of Mouse Bone Marrow (Day 0) 3.2 Ex Vivo Differentiation and Culture of Eos from Adult Mouse Bone Marrow 3.3 The Murine AEU: Co-culture of BMMCs and BMEos 3.4 Activation of the AEU (BMMCs/BMEos) 3.5 Mediator Assays 3.5.1 Tryptase Assay 3.5.2 Assay for Measuring Released β-Hex (See also Note 9) 3.5.3 Assay for Measuring Released EPO from Eos (See also Note 10) 3.5.4 Cytokines and Chemokines 3.6 The Human AEU: Co-culture of pbEos and CBMCs 3.7 Other Options of Co-culture 3.7.1 Transwell Co-culture 3.7.2 Blocking the Receptor on the Cells 3.7.3 Knockout Cell Co-Culture 4 Notes References Chapter 19: Assessing Phenotypic Heterogeneity in Intestinal Tissue Eosinophils 1 Introduction 2 Materials 2.1 Additional Materials and Equipment 2.2 Buffers and Other Reagents for Intestinal Cell Isolations (See Note 1) 2.3 Flow Cytometry Reagents 3 Methods 3.1 Harvesting Intestine and Removal of Peyer´s Patches 3.2 Isolation of Intraepithelial (IE) Leukocytes 3.3 Isolation of Lamina Propria (LP) Leukocytes 3.4 Density Gradient Purification of Leukocytes 3.5 Flow Cytometry of Intestinal Leukocytes 4 Notes References Chapter 20: Antimicrobial Activity of Human Eosinophil Granule Proteins 1 Introduction 2 Materials 2.1 Culture Media and Buffers 2.2 Shell Vial Assay 2.3 Antibody and Conjugate 2.4 RT-PCR Assay for Antiviral Activity Against RSV 2.5 Assay for Antihelminthic Activity Against S. mansoni 2.6 Assay of Antiprotozoan Activity Against L. donovani 2.7 Assay of Antibacterial Activity Against E. coli 2.8 Antifungal Activity Against Candida albicans 2.9 Biofilm Formation and Eradication 3 Methods 3.1 Assay of Antiviral Activity Against RSV 3.1.1 Shell Vial Assay for Antiviral Activity 3.1.2 RT-PCR-Based Assay for Antiviral Activity Against RSV 3.2 Assay of Antihelminthic Activity Against S. mansoni 3.3 Assay of Antiprotozoan Activity Against Leishmania donovani UR6 3.4 Assay of Antibacterial Activity Against E. coli 3.5 Assay of Antifungal Activity Against Candida albicans 3.6 Assay of Anti-Biofilm Activity 4 Notes References Chapter 21: Experimental Modeling of Eosinophil-Associated Diseases 1 Introduction 2 Materials 2.1 Experimental Model of Aspergillus fumigatus-Induced Eosinophilic Asthma 2.2 Experimental Models of OVA Allergen-Induced Eosinophilic Asthma/Food-Allergen-Induced EoE/Gastroenteritis/Eosinophilic Col... 2.3 Tissue Eosinophil Analysis 2.4 Anti-MBP Immunostaining of Tissue Sections 3 Methods 3.1 Experimental Model of Aspergillus fumigatus-Induced Eosinophilic Asthma 3.2 Experimental Model of OVA Allergen-Induced Eosinophilic asthma (See Fig. 2) 3.3 Flow Cytometry Analysis of Eosinophils 3.4 Tissue Eosinophil Analysis (H&E Staining) 3.5 Anti-MBP Immunostaining of Tissue Sections 3.5.1 Mouse Model of Eosinophilic Gastrointestinal Disorders (EGID) 3.5.2 Mouse Model of Aeroallergen (Aspergillus fumigatus)-Induced Eosinophilic Esophagitis (EoE) 3.6 Mouse Model of Food Allergen-Induced EoE and Gastroenteritis (See Fig. 7) 3.7 Mouse Model of Eosinophilic Colitis (See Fig. 9) 3.8 Mouse Model of Cerulein-Induced Eosinophilic Pancreatitis (See Fig. 11) 4 Notes References Index