Dendritic Cell Protocols (Methods in Molecular Biology, 1423)

✍ Scribed by Elodie Segura (editor), Nobuyuki Onai (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 317
• Category: Library

✦ Synopsis

The third edition of this volume is aimed at providing both beginners and more experienced researchers a choice of methods to isolate and analyze dendritic cells(DC). An introductory review provides an overview of recent advances in the characterization of DC subsets in mouse and human. While additional chapters provide methods to culture human and mouse dendritic cells, protocols for the isolation of dendritic cells, the isolation of dendritic cell progenitors from mouse, and the purification of dendritic cells from human blood. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Dendritic Cell Protocols, Third Edition aims to ensure successful results in the further study of this vital field.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Introduction
Chapter 1: Review of Mouse and Human Dendritic Cell Subsets
1 Introduction
2 Mouse Dendritic Cell Subsets
2.1 Resident Versus Migratory DCs
2.2 Plasmacytoid DCs
2.3 Classical DCs
2.3.1 Batf3-
2.3.2 IRF4-­Dependent DCs
2.4 Langerhans Cells
2.5 Monocyte-
3 Human Dendritic Cell Subsets
3.1 Blood Versus Tissue DCs
3.2 Plasmacytoid DCs
3.3 Classical DCs
3.4 CD1a+ and CD14+ Tissue DCs
3.5 Langerhans Cells
3.6 Monocyte-
References
Part II: In Vitro Culture of Dendritic Cells
Chapter 2: In Vitro Generation of Human XCR1+ Dendritic Cells from CD34+ Hematopoietic Progenitors
1 Introduction
2 Materials
2.1 Enrichment of CD34+ Hematopoietic Progenitor Stem Cells
2.2 Expansion of Hematopoietic Precursors
2.3 Cryopreservation and Revival of Expanded Hematopoietic Precursors
2.4 Differentiation of DCs from Expanded Hematopoietic Precursors
2.5 Staining for Flow Cytometry Analysis
2.6 Sort of DC Subsets at the End of the Culture
2.7 Stimulation of Sorted DC Subsets with Different Adjuvants
3 Methods
3.1 Isolation of the Mononuclear Cells (MNCs) from Cord Blood, Bone Marrow Aspirate, or G-CSF-Mobilized Peripheral Blood Apheresis
3.2 Enrichment of CD34+ Hematopoietic Progenitor Stem Cells
3.3 Expansion of Hematopoietic Precursors
3.4 Cryopreservation of Expanded Hematopoietic Precursors
3.5 Revival of Frozen Expanded Hematopoietic Precursors
3.6 Differentiation of DCs from Expanded Hematopoietic Precursors
3.7 Phenotypic Identification of the Different Cell Populations at the End of the Culture
3.8 Sorting of the DC Subsets at the End of the Differentiation Culture
3.9 Stimulation of Sorted DC Subsets with Different Adjuvants and Evaluation of Their Activation
4 Notes
References
Chapter 3: Derivation and Utilization of Functional CD8+ Dendritic Cell Lines
1 Introduction
2 Materials
2.1 Derivation of CD8+ DC Lines
2.2 Freezing
2.3 Cell Culture
2.4 Lentiviral Transduction
3 Methods
3.1 Derivation of CD8+ DC Lines from Tumor-­Bearing Mushi Mice
3.2 Freezing MuTu DC Lines
3.3 Culture of MuTu DC Lines
3.4 Lentiviral Transduction
4 Notes
References
Part III: Purification and Isolation of Dendritic Cell Subsets
Chapter 4: Isolation of Dendritic Cell Progenitor and Bone Marrow Progenitor Cells from Mouse
1 Introduction
2 Materials
2.1 Preparation of Bone Marrow Cell (BMCs)
2.2 Isolation of Lineage Negative Cells from BMCs
2.3 Antibody Staining and Cell Sorting for DC and BM Progenitors
2.4 Antibody Staining and Cell Sorting for Pre-cDC
3 Methods
3.1 Preparation of Bone Marrow Cells (BMCs)
3.2 Isolation of Lineage-­Negative Cells from BMCs
3.3 Antibody Staining and Cell Sorting for DC and BM Progenitors
3.4 Antibody Staining and Cell Sorting for Pre-cDC
4 Notes
References
Chapter 5: The Isolation and Enrichment of Large Numbers of Highly Purified Mouse Spleen Dendritic Cell Populations and Their In Vitro Equivalents
1 Introduction
2 Materials
2.1 In Vivo Administration of Flt3L
2.2 Organ Removal
2.3 Digestion of Spleen and Release of DC
2.4 Selection of the Light-­Density Fraction of Spleen
2.5 Depletion of Non-DC Lineages
2.6 In Vitro Administration of Flt3L
2.6.1 Immunofluorescent Staining and Purification via Flow Cytometric Sorting
2.6.2 Immunofluorescent Staining and Purification via Immunomagnetic Beads
2.6.3 Staining and Purification via Immunomagnetic Beads: Removal of NK Cells
3 Methods
3.1 In Vivo Administration of Flt3L
3.1.1 Soluble Flt3L
3.1.2 B16Flt3L
3.2 Organ Removal
3.3 Digestion of Spleen and Release of DC
3.4 Selection of the Light-­Density Fraction of Spleen
3.5 Depletion of Non-DC Lineages
3.6 In Vitro Administration of Flt3L
3.7 Immuno-­fluorescent Staining and Purification
3.7.1 Via Flow Cytometric Sorting
3.7.2 Via Immunomagnetic Beads
3.7.3 Depletion of NK Cells via Immunomagnetic Beads
4 Notes
References
Chapter 6: Dendritic Cell Subset Purification from Human Tonsils and Lymph Nodes
1 Introduction
2 Materials
2.1 Mechanical and Enzymatic Tissue Digestion
2.2 DC Enrichment
2.3 Cell Sorting of the DC Subsets
3 Methods
3.1 Purification of DC from Tonsils
3.1.1 Mechanical and Enzymatic Tissue Digestion
3.1.2 DC Enrichment
3.1.3 Cell Sorting of the DC Subsets
3.2 Purification of DC from Lymph Nodes
3.2.1 Mechanical and Enzymatic Tissue Digestion
3.2.2 DC Enrichment
3.2.3 Cell Sorting of the DC Subsets
4 Notes
References
Chapter 7: Isolation and Identification of Conventional Dendritic Cell Subsets from the Intestine of Mice and Men
1 Introduction
2 Materials
2.1 Reagents (See Note 1)
2.2 Equipment
3 Methods
3.1 Isolation of Leukocytes from Mouse Small Intestinal Lamina Propria
3.2 Isolation of Leukocytes from Mouse Colonic Lamina Propria
3.3 Isolation of Leukocytes from Human Colonic and Small Intestinal Biopsies
3.4 Isolation of Leukocytes from Resected Human Intestine
3.5 Preparation of Cells for FACS Analysis
3.6 Phenotypic Identification of Conventional DCs in the Intestine
3.6.1 Identifying DCs in the Murine Intestine
3.6.2 DCs in the Human Intestine
4 Notes
References
Chapter 8: Isolation of Human Skin Dendritic Cell Subsets
1 Introduction
2 Materials
2.1 Dermatome-Cut Skin Preparation
2.2 Single-Cell Suspension Preparation
2.3 Immunostaining and Cell Sorting
3 Methods
3.1 Epidermis and Dermis Isolation
3.2 Single-Cell Suspension Preparation
3.3 Immunostaining and Cell Sorting
3.4 Identification of Skin DCs and Macrophage Subsets
4 Notes
References
Chapter 9: Isolation of Mouse Dendritic Cell Subsets and Macrophages from the Skin
1 Introduction
2 Materials
3 Methods
3.1 Tissues Collection
3.2 Enzymatic Treatment
3.3 Percoll Gradient (See Note 3)
3.4 FACS Staining (See Note 4)
3.5 Gating Strategy for FACS Analysis or Cell Sorting
4 Notes
References
Chapter 10: Isolation of Conventional Dendritic Cells from Mouse Lungs
1 Introduction
2 Materials
2.1 Anesthesia Solution
2.2 Buffers
2.3 Media and Supplements
2.4 Digestion Medium
2.5 Enrichment
2.6 Flow Cytometry
2.7 Equipment
3 Methods
3.1 Anesthetization of Mice
3.2 Dissection
3.3 Digestion
3.4 Enrichment
3.4.1 Enrichment Through Positive Selection with CD11c+ Microbeads
3.4.2 Enrichment Through Negative Selection of Lineage− Cells
3.4.3 Enrichment Through OptiPrep
3.5 Cell Staining and Sorting
4 Notes
References
Chapter 11: Purification of Human Dendritic Cell Subsets from Peripheral Blood
1 Introduction
2 Materials
2.1 Total PBMCs Purification from Blood
2.2 Total DC Enrichment from PBMCs
2.3 Immunofluo­rescence Staining and FACS
3 Methods
3.1 Total PBMCs Purification from Blood
3.2 Total DC Enrichment from PBMCs
3.3 Immunofluo­rescence Staining and FACS
4 Notes
References
Chapter 12: Protocols for the Identification and Isolation of Antigen-­Presenting Cells in Human and Mouse Tissues
1 Introduction
2 Materials
2.1 Human Tissue Processing
2.2 Mouse Tissue Processing
3 Methods
3.1 Human Tissue Processing
3.1.1 Isolation of Antigen-­Presenting Cells from Human Skin
3.1.2 Enrichment of Antigen-­Presenting Cells from Human Soft Tissues Including the Lung, Spleen, and Liver
3.1.3 Flow Cytometry of Antigen-­Presenting Cells
3.1.4 Whole Mount Microscopy of Human Dermis
3.2 Mouse Tissue Processing
3.2.1 Murine Lung, Liver, and Kidney Tissue Processing
3.2.2 Enrichment of the Lamina Propria and Intra-­Epithelial Antigen-Presenting Cells from Murine Small Intestine and Colon
3.2.3 Enrichment of Antigen-­Presenting Cells from Mouse Ear Skin
3.2.4 Flow Cytometry Analysis of Murine Antigen-­Presenting Cells
4 Notes
References
Part IV: Ex Vivo Functional Analysis of Dendritic Cells
Chapter 13: Measurement of Export to the Cytosol in Dendritic Cells Using a Cytofluorimetry-Based Assay
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 14: Cross-Presentation Assay for Human Dendritic Cells
1 Introduction
2 Materials
2.1 Cross-­Presentation Assay
2.2 Induction of Necrosis in Antigen-­Expressing Cells
2.3 Assessment of Antigen-­Specific T Cell Clone Activation
2.3.1 Analysis of IFNγ Secretion by ELISA
2.3.2 Analysis of IFNγ Secretion by Intracellular Flow Cytometry
3 Methods
3.1 Cross-­Presentation of Soluble Antigen
3.2 Cross-­Presentation of Necrotic Cell-Associated Antigen
3.3 Assessment of T Cell Clone Activation
3.3.1 ELISA
3.3.2 Intracellular Staining
4 Notes
References
Chapter 15: Analysis of Intracellular Trafficking of Dendritic Cell Receptors for Antigen Targeting
1 Introduction
2 Materials
2.1 FIP Labeling
2.2 SHIP Assay
3 Methods
3.1 FIP Labeling
3.2 SHIP Assay (See Note 9)
3.3 Data Analysis
4 Notes
References
Chapter 16: Characterization of Dendritic Cell Subsets Through Gene Expression Analysis
1 Introduction
2 Materials
2.1 Accessing the Expression Dataset for the First Tutorial
2.2 Accessing the Expression Dataset for the Second Tutorial
2.3 Preprocessing ofthe Data and Various Statistical Analyses
2.4 Classification by Hierarchical Clustering
2.5 Handling of the Expression Datasets
2.6 Data Reduction by Principal Component Analysis
2.7 Gene Set-Based Approaches
3 Methods
3.1 Normalization and Background Correction of the Data for the First Tutorial
3.2 Quality Control Analyses of the Data for the First Tutorial
3.3 Classification of the Data of the First Tutorial by Hierarchical Clustering
3.3.1 Classification by Hierarchical Clustering on All Probe Sets
3.3.2 Remove the Probes Always Below the Background Level
3.3.3 Remove the Probes with No or Little Expression Changes Across Cell Populations
3.3.4 Apply a Multiscale Bootstrap Resampling to Hierarchical Clustering
3.4 Data Reduction by Principal Component Analysis on the Data for the First Tutorial
3.5 Gene Set-Based Approach Applied to the Data for the First Tutorial
3.5.1 Generate Cell-Specific Transcriptomic Signatures
3.5.2 Assess the Enrichment of the Transcriptomic Signatures
3.6 Computational Analysis of the Data for the Second Tutorial
4 Notes
References
Part V: In Vivo Analysis of Dendritic Cells
Chapter 17: In Vivo Ablation of a Dendritic Cell Subset Expressing the Chemokine Receptor XCR1
1 Introduction
2 Materials
2.1 Mice
2.2 Reagents
3 Materials
4 Methods
4.1 Ablation of XCR1+ Cells in XCR1-­DTRvenus Mice
4.2 Evaluation of Deletion Efficiency
5 Notes
References
Chapter 18: In Vivo Analysis of Intestinal Mononuclear Phagocytes
1 Introduction
2 Materials
2.1 Animals
2.2 Reagents
2.3 Materials
3 Methods
3.1 In Vivo Ablation of Intestinal Mononuclear Phagocytes
3.1.1 Conditional Short-Term Ablation of CD11chigh DC
3.1.2 Conditional Long-Term Ablation of CD11chigh DC
3.1.3 Conditional Long-Term Ablation of Mutated DC Compartments
3.2 Adoptive Precursor Transfers into Mononuclear Phagocyte-Depleted Mice
3.3 Isolation of Intestinal Lamina Propria Mononuclear Phagocytes
4 Notes
References
Chapter 19: In Vivo Imaging of Cutaneous DCs in Mice
1 Introduction
2 Materials
2.1 Reagents and Equipment
2.2 Instruments
2.3 Animals
3 Methods
3.1 Labeling of Cutaneous DCs
3.2 Preimaging Preparations
3.3 Placing the Mouse onto the Microscope Stage
3.4 Observation of Cutaneous DCs
4 Notes
References
Chapter 20: Analysis of Dendritic Cell Function Using Clec9A-DTR Transgenic Mice
1 Introduction
1.1 Dendritic Cells (DCs), a Heterogeneous Family of Myeloid Cells
1.2 Clec9A Expression Profile and Function
1.3 DTR-Mediated DC Ablation in Transgenic Mice
1.4 Experimental Cerebral Malaria (ECM) in Clec9A-­DTR Mice
2 Materials
2.1 Conditional Depletion of CD8/CD103+ DCs in Clec9A-­DTR Mice
2.2 Isolation of DCs from Lymphoid and Nonlymphoid Tissues
2.3 Analysis of DC Depletion in Lymphoid and Nonlymphoid Organs
2.4 In Vivo Analysis of CD8+ DC Function in ECM Model
3 Methods
3.1 Conditional Depletion of CD8/CD103+ DCs in Clec9A-­DTR Mice
3.2 Isolation of DCs from Lymphoid and Nonlymphoid Tissues
3.3 Analysis of DC Depletion in Lymphoid and Nonlymphoid Organs of DT-Treated Clec9A DTR Mice
3.4 In Vivo Analysis of CD8+ DC Function in Experimental Cerebral Malaria Model (ECM)
4 Notes
References
Chapter 21: Analysis of DC Functions Using CD205-DTR Knock-In Mice
1 Introduction
2 Materials
2.1 Mice
2.2 Generation of Bone Marrow Chimeric Mice and DT Treatment
2.3 Assessment of Depletion Efficiency
2.4 OVA Cross Presentation Assay
2.5 CTL Generation Assay
2.6 Assay for Antibacterial Immunity
3 Methods
3.1 Generation of Bone Marrow (BM) Chimeric Mice and DT Treatment
3.1.1 Preparation of Single-Cell Suspensions from the Bone Marrow (BM)
3.1.2 Generation of BM Chimeric Mice
3.1.3 DT Treatment
3.2 Assessment of Depletion Efficiency
3.2.1 Preparation of Single-Cell Suspensions from the Spleen of DT-Treated Mice
3.2.2 Purification of CD11c+ DCs from Single-Cell Suspensions
3.2.3 Analysis of DC Depletion Efficiency by Flow Cytometry
3.3 OVA Cross Presentation Assay In Vivo
3.3.1 Preparation of Single-Cell Suspensions from the Spleen of OT-I Mice
3.3.2 Purification of CD8+ T Cells from Single-Cell Suspensions
3.3.3 CFSE Labeling of OT-I T Cells
3.3.4 Adoptive Transfer and Immunization
3.3.5 Analysis of CD8+ T-Cell Proliferation by Flow Cytometry
3.4 CTL Generation Assay
3.4.1 Immunization
3.4.2 Tetramer Staining of CD8+ T Cells
3.4.3 Intracellular Staining for CD8+IFN-γ+ T Cells
3.5 Assay for Antibacterial Immunity
3.5.1 Bacterial Infection
3.5.2 Determination of Bacterial Burden
4 Notes
References
Chapter 22: Generation of Humanized Mice for Analysis of Human Dendritic Cells
1 Introduction
2 Materials
2.1 Isolation of CD34+ Hematopoietic Stem and Progenitor Cells
2.2 Intrahepatic Injection of Human CD34+ Cells
2.3 FACS Analysis of Human Dendritic Cells
3 Methods
3.1 Isolation of CD34+ Hematopoietic Stem and Progenitor Cells
3.1.1 Isolation of Mononuclear Cells from Cytokine-­Mobilized Peripheral Blood
3.1.2 Magnetic Labeling and Separation
3.2 Intrahepatic Injection of Human CD34+ Cells
3.2.1 Finger Cutting and Genotyping
3.2.2 Irradiation of Newborn Mice
3.2.3 Preparation of CD34+ Cells
3.2.4 Cell Injection for Transplantation
3.3 FACS Analysis of Human Dendritic Cells from Humanized Mice
3.3.1 Preparation of Cell Suspension from Peripheral Blood
3.3.2 Preparation of Cell Suspension from the Bone Marrow
3.3.3 Preparation of Cell Suspension from the Spleen and Thymus
3.3.4 Preparation of Cell Suspension from the Liver and Lung
3.4 Antibody Staining
4 Notes
References
Index