Current Protocols in Immunology

Current Protocols in Immunology

Online ISBN: 9780471142737

DOI: 10.1002/0471142735

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  1. Foreword
  2. Preface
  3. Chapter 1 Care and Handling of Laboratory Animals
    1. Introduction
    2. Section I Animal Health and Husbandry
      1. Unit 1.1 Animal Health Assurance
      2. Unit 1.2 Managing Immunocompromised Animals
      3. Unit 1.3 Handling and Restraint
      4. Unit 1.4 Anesthesia
      5. Unit 1.5 Animal Identification
    3. Section II Use of Mouse, Rat, Hamster, and Rabbit
      1. Unit 1.6 Parenteral Injections
      2. Unit 1.7 Blood Collection
      3. Unit 1.8 Euthanasia
    4. Section III Selected Surgical Procedures
      1. Unit 1.9 Removal of Lymphoid Organs
      2. Unit 1.10 Survival Surgery: Removal of the Spleen or Thymus
      3. Unit 1.11 Surgical Removal of Mouse Salivary Glands
      4. Unit 1.12 Techniques in Aseptic Rodent Surgery
  4. Chapter 2 Induction of Immune Responses
    1. Introduction
    2. Section I Assays for Antibody Production
      1. Unit 2.1 Enzyme-Linked Immunosorbent Assays
      2. Unit 2.2 Isotype Determination of Antibodies
      3. Unit 2.3 Double-Immunodiffusion Assay for Detecting Specific Antibodies
    3. II Production of Antibodies
      1. Production of Antibodies
      2. Unit 2.4 Production of Polyclonal Antisera
      3. Unit 2.5 Production of Monoclonal Antibodies
      4. Unit 2.6 Monoclonal Antibody Supernatant and Ascites Fluid Production
    4. Section III Purification and Fragmentation of Antibodies
      1. Unit 2.7 Purification of Immunoglobulin G
      2. Unit 2.8 Fragmentation of Immunoglobulin G
      3. Unit 2.9 Isolation of Murine and Human Immunoglobulin M and Murine Immunoglobulin D
      4. Unit 2.9B Measurement of Human Serum IgD Levels
      5. Unit 2.10A Fragmentation of Immunoglobulin M
      6. Unit 2.10B Purification of Human IgA
    5. Section IV Specialized Techniques
      1. Unit 2.11 Preparation of Immune Stimulating Complexes (ISCOMs) as Adjuvants
      2. Unit 2.12 Cloning, Expression, and Modification of Antibody V Regions
      3. Unit 2.13 Production of Bispecific Antibodies
      4. Unit 2.14 Nucleic Acid Immunizations
      5. Unit 2.15 Vaccination of Mice with Baculovirus-Infected Insect Cells Expressing Antigenic Proteins
      6. Unit 2.16 Efficient Delivery of Antibody Into Living Cells Using Hemagglutinating Virus of Japan (HVJ) Envelope
  5. Chapter 3 In Vitro Assays for Mouse Lymphocyte Function
    1. Introduction
    2. Section I Isolation and Fractionation of Mononuclear Cell Populations
      1. Unit 3.1 Isolation of Mouse Mononuclear Cells
      2. Unit 3.2 T Cell Enrichment By Nonadherence to Nylon
      3. Unit 3.3 T Cell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells
      4. Unit 3.4 T Cell Depletion by Cytotoxic Elimination
      5. Unit 3.5A Fractionation of T and B Cells Using Magnetic Beads
      6. Unit 3.5B Isolation of Ly-1+/CD5+ B Cells by Cell Sorting
      7. Unit 3.6 Depletion of Accessory Cells by Adherence to Sephadex G-10
      8. Unit 3.7 Isolation of Dendritic Cells
    3. Section II Assays for B Cell Function
      1. Unit 3.8 Assays for B Lymphocyte Function
      2. Unit 3.9 Early Events in B Lymphocyte Activation
      3. Unit 3.10 Proliferative Assays for B Cell Function
    4. Section III Assays for T Cell Function
      1. Unit 3.11 Induction and Measurement of Cytotoxic T Lymphocyte Activity
      2. Unit 3.12 Proliferative Assays for T Cell Function
      3. Unit 3.13 Production of TH1 and TH2 Cell Lines and Clones
      4. Unit 3.14 Production of Mouse T Cell Hybridomas
      5. Unit 3.15 Quantitation of Functional T Cells by Limiting Dilution
      6. Unit 3.16 Granule Enzyme Exocytosis Assay for Cytotoxic T Lymphocyte Activation
    5. Section IV Related Isolation Procedures and Functional Assays
      1. Unit 3.17 Measurement of Apoptosis and Other Forms of Cell Death
      2. Unit 3.18 In Vitro Systems for the Study of T Cell Development: Fetal Thymus Organ Culture and OP9-DL1 Cell Coculture
      3. Unit 3.19 Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes, Peyer's Patch, and Lamina Propria Cells
      4. Unit 3.20 Isolation of Mouse Neutrophils
      5. Unit 3.21 Isolation of Mouse Intrahepatic Lymphocytes
      6. Unit 3.22 Isolation of Murine Natural Killer Cells
      7. Unit 3.23 Generation, Isolation, and Maintenance of Rodent Mast Cells and Mast Cell Lines
  6. Chapter 4 In Vivo Assays for Lymphocyte Function
    1. Introduction
    2. Unit 4.1 In Vivo Depletion of CD4- and CD8-Specific T Cells
    3. Unit 4.2 Contact Hypersensitivity
    4. Unit 4.3 Animal Models of Acute and Chronic Graft-Versus-Host Disease
    5. Unit 4.4 Skin Allograft Rejection
    6. Unit 4.5 Delayed-Type Hypersensitivity
    7. Unit 4.6 Assessment of Lymphocyte Development in Radiation Bone Marrow Chimeras
    8. Unit 4.7 Measurement of T and B Cell Turnover with Bromodeoxyuridine
    9. Unit 4.8 Construction of Human-SCID Chimeric Mice
    10. Unit 4.9 Use of the Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation
  7. Chapter 5 Immunofluorescence and Cell Sorting
    1. Introduction
    2. Unit 5.1 Overview of Flow Cytometry
    3. Unit 5.2 Analysis of Flow Cytometry Data
    4. Unit 5.3 Preparation of Cells and Reagents for Flow Cytometry
    5. Unit 5.4 Flow Cytometry Analysis Using the Becton Dickinson FACS Calibur
    6. Unit 5.5 Measurement of Intracellular Ions by Flow Cytometry
    7. Unit 5.6 Measurement of Intercellular Conjugates by Flow Cytometry
    8. Unit 5.7 Analysis of Cellular DNA Content by Flow Cytometry
    9. Unit 5.8 Multiparameter FACS Analysis
    10. Unit 5.9 Visualization of Multiprotein Complexes by Flow Cytometry
  8. Chapter 6 Cytokines and Their Cellular Receptors
    1. Introduction
    2. Unit 6.1 Measurement of Lymphokine Receptors
    3. Unit 6.2 Measurement of Soluble and Membrane-Bound Interleukin 1 Using a Fibroblast Bioassay
    4. Unit 6.3 Measurement of Human and Murine Interleukin 2 and Interleukin 4
    5. Unit 6.4 Measurement of Interleukin 3 and Other Hematopoietic Cytokines, such as GM-CSF, G-CSF, M-CSF, Erythropoietin, Steel Factor, and Flt-3 Ligand
    6. Unit 6.5 Measurement of Mouse and Human Interleukin 5
    7. Unit 6.6 Measurement of Interleukin 6
    8. Unit 6.7 Measurement of Interleukin 7
    9. Unit 6.8 Measurement of Mouse and Human Interferon gamma
    10. Unit 6.9 Measurement of Antiviral Activity Induced by Interferons alpha, beta, and gamma
    11. Unit 6.10 Measurement of Tumor Necrosis Factor alpha and beta
    12. Unit 6.11 Measurement of TGF-beta in Biological Fluids
    13. Unit 6.12 Biological Responses to Chemokine Superfamily Members
    14. Unit 6.13 Measurement of Mouse and Human Interleukin 9
    15. Unit 6.14 Measurement of Mouse and Human Interleukin 10
    16. Unit 6.15 Measurement of Human Interleukin 11
    17. Unit 6.16 Measurement of Human and Mouse Interleukin-12
    18. Unit 6.17 Measurement of Human and Murine Stem Cell Factor (c-kit Ligand)
    19. Unit 6.18 Measurement of Interleukin-13
    20. Unit 6.19 ELISPOT Assay to Detect Cytokine-Secreting Murine and Human Cells
    21. Unit 6.20 Immunoenzymetric Assay of Mouse and Human Cytokines Using NIP-Labeled Anti-Cytokine Antibodies
    22. Unit 6.21 Detection of Cytokine Receptors by Flow Cytometry
    23. Unit 6.22 Measurement of Interleukin 15
    24. Unit 6.23 Measurement of Interleukin 16
    25. Unit 6.24 Detection of Intracellular Cytokines by Flow Cytometry
    26. Unit 6.25 Measurement of Interleukin-17
    27. Unit 6.26 Measurement of Human and Mouse Interleukin 18
    28. Unit 6.27 Detection and Isolation of Cytokine Secreting Cells Using the Cytometric Cytokine Secretion Assay
    29. Unit 6.28 The In Vivo Cytokine Capture Assay for Measurement of Cytokine Production in the Mouse
    30. Unit 6.29 Cytokine, Growth Factor, and Chemokine Ligand Database
    31. Unit 6.30 Measurement of Interleukin-21
  9. Chapter 7 Immunologic Studies in Humans
    1. Introduction
    2. I Preparation of Human Mononuclear Cell Populations and Subpopulations
      1. I Preparation of Human Mononuclear Cell Populations and Subpopulations
      2. Unit 7.1 Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood
      3. Unit 7.2 Isolation of T Cells Using Rosetting Procedures
      4. Unit 7.3 Purification of T Cell Subpopulations
      5. Unit 7.4 Immunomagnetic Purification of T Cell Subpopulations
      6. Unit 7.4B The Purification and Functional Analysis of Human CD4+CD25high Regulatory T Cells
      7. Unit 7.5 Isolation of Human B Cell Populations
      8. Unit 7.6A Isolation of Human Monocyte Populations
      9. Unit 7.6B Isolation and Purification of Human Intestinal Macrophages
      10. Unit 7.7 Isolation of Human NK Cells and Generation of LAK Activity
      11. Unit 7.8 Isolation of Mononuclear Cells from Tonsillar Tissue
      12. Unit 7.9 Detection of Unseparated Human Lymphocytes by Flow Cytometry
    3. II Measurement of Basic Immunologic Characteristics of Human Mononuclear Cells
      1. Measurement of Basic Immunologic Characteristics of Human Mononuclear Cells
      2. Unit 7.10 Measurement of Proliferative Responses of Cultured Lymphocytes
      3. Unit 7.11 Induction of Polyclonal Immunoglobulin Synthesis
      4. Unit 7.12 Measurement of Polyclonal Immunoglobulin Synthesis Using ELISA
      5. Unit 7.13 Measurement of Polyclonal Immunoglobulin Synthesis Using the Reverse Plaque Technique
      6. Unit 7.14 Measurement of Immunoglobulin Synthesis Using the ELISPOT Assay
      7. Unit 7.15 Measurement of In Vitro Specific-Antibody Synthesis
      8. Unit 7.16 Induction and Measurement of In Vivo Antibody Responses
      9. Unit 7.17 Measurement of Polyclonal and Antigen-Specific Cytotoxic T Cell Function
      10. Unit 7.18 Measurement of Cytotoxic Activity of NK/LAK Cells
      11. Unit 7.18B Measurement of Cytokine Production Using Whole Blood
    4. Section III Long-Term and Immortalized Human Cell Lines and Clones
      1. Unit 7.19 Generation and Maintenance of Cloned Human T Cell Lines
      2. Unit 7.20 Generation of HTLV-I-Transformed T Cell Lines
      3. Unit 7.21 Herpesvirus saimiri Transformation of Human T Lymphocytes
      4. Unit 7.21B Immortalization of Human and Rhesus Macaque Primary Antigen-Specific T Cells by Retrovirally Transduced Telomerase Reverse Transcriptase
      5. Unit 7.22 Generation of Epstein-Barr Virus (EBV)–Immortalized B Cell Lines
    5. Section IV Preparation and Functional Analysis of Other Human Lymphoid and Nonlymphoid Cells
      1. Unit 7.23 Isolation and Functional Analysis of Neutrophils
      2. Unit 7.24 Isolation of Human Basophils
      3. Unit 7.25 Isolation of Tissue Mast Cells
      4. Unit 7.26 Measuring Degranulation of Mast Cells
      5. Unit 7.27 51Cr Release Assay of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
      6. Unit 7.28 Measurement of Cellular Adhesion Under Static Conditions
      7. Unit 7.29 Cell Adhesion Under Hydrodynamic Flow Conditions
      8. Unit 7.30 Isolation of Human Intestinal Mucosal Mononuclear Cells
      9. Unit 7.31 Isolation of Human Eosinophils
      10. Unit 7.32 Isolation and Generation of Human Dendritic Cells
      11. Unit 7.33 Measurement of Prostaglandins and Other Eicosanoids
      12. Unit 7.34 Isolation and Characterization of Human Natural Killer Cell Subsets
      13. Unit 7.35 Assays for B Cell and Germinal Center Development
      14. Unit 7.36 In Vitro Models of Human T Cell Anergy
      15. Unit 7.37 Generation, Isolation, and Maintenance of Human Mast Cells and Mast Cell Lines Derived from Peripheral Blood or Cord Blood
      16. Unit 7.38 Measuring Mast Cell Mediator Release
      17. Unit 7.39 Evaluation of Human Natural Killer Cell Activities in Whole Blood
      18. Unit 7.40 Isolation of Cells from the Feto-Maternal Interface
  10. Chapter 8 Isolation and Analysis of Proteins
    1. Introduction
    2. Section I Solubilization of Cellular Proteins
      1. Unit 8.1A Solubilization of Lymphocytes
      2. Unit 8.1B Purification of Organelles from Mammalian Cells
      3. Unit 8.1C Subcellular Fractionation of Tissue Culture Cells
    3. Section II Isolation of Proteins Using Antibodies
      1. Unit 8.2 Immunoaffinity Chromatography
      2. Unit 8.3 Immunoprecipitation
    4. Section III Electrophoretic Separation of Proteins
      1. Unit 8.4 One-Dimensional SDS Gel Electrophoresis of Proteins
      2. Unit 8.5 Two-Dimensional Gel Electrophoresis
      3. Unit 8.6 Diagonal Gel Electrophoresis
      4. Unit 8.7 Detection of Protein-Protein Interactions by Coprecipitation
      5. Unit 8.8 Electroelution of Proteins from Stained Gels
    5. Section IV Detection of Proteins
      1. Unit 8.9 Staining Proteins in Gels
      2. Unit 8.10 Immunoblotting and Immunodetection
      3. Unit 8.10B Detection of Proteins on Blot Transfer Membranes
      4. Unit 8.11 Iodination of Soluble and Membrane-Bound Proteins
      5. Unit 8.12 Biosynthetic Labeling of Proteins
      6. Unit 8.13 Metabolic Radiolabeling of Animal Cell Glycoconjugates
      7. Unit 8.14 Inhibition of N-Linked Glycosylation
      8. Unit 8.15 Endoglycosidase and Glycoamidase Release of N-Linked Glycans
      9. Unit 8.16 Biotinylation and Analysis of Membrane-Bound and Soluble Proteins
      10. Unit 8.17 Single-Cell Phospho-Protein Analysis by Flow Cytometry
  11. Chapter 9 Peptides
    1. Introduction
    2. Unit 9.1 Introduction to Peptide Synthesis
    3. Unit 9.2 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins
    4. Unit 9.3 Production of Anti-Peptide Antisera
    5. Unit 9.4 B Cell Epitope Mapping Using Synthetic Peptides
    6. Unit 9.5 Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries
    7. Unit 9.6 Synthesis and Application of Peptide Dendrimers As Protein Mimetics
    8. Unit 9.7 Synthesis of Multiple Peptides on Plastic Pins
    9. Unit 9.8 Phage Display Selection, Analysis, and Prediction of B Cell Epitopes
    10. Unit 9.9 Epitope Mapping Using Gram-Positive Surface Display
  12. Chapter 10 Molecular Biology
    1. Introduction
    2. Section I Isolation and Purification of DNA
      1. Unit 10.1 Purification and Concentration of DNA from Aqueous Solutions
      2. Unit 10.2 Preparation of Genomic DNA from Mammalian Tissue
      3. Unit 10.3 Preparation of Bacterial Plasmid DNA
    3. Section II Resolution and Recovery of DNA Fragments
      1. Unit 10.4 Agarose Gel Electrophoresis
      2. Unit 10.5 Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
    4. Section III Analysis of Genomic DNA by Blotting and Hybridization
      1. Unit 10.6A Southern Blotting
      2. Unit 10.6B Hybridization Analysis of DNA Blots
      3. Unit 10.7 Deprotection of Oligonucleotides and Purification Using Denaturing PAGE
    5. Section IV Enzymatic Cleavage and Labeling of DNA
      1. Unit 10.8 Digestion of DNA with Restriction Endonucleases
      2. Unit 10.9 Reagents and Radioisotopes Used to Manipulate Nucleic Acids
      3. Unit 10.10 Enzymatic Labeling of Nucleic Acids
    6. Section V Preparation and Analysis of RNA
      1. Unit 10.11 Preparation of RNA from Tissues and Cells
      2. Unit 10.12 Analysis of RNA by Northern and Slot-Blot Hybridization
    7. Section VI Transfection and Expression of Cloned DNA
      1. Unit 10.13 Calcium Phosphate Transfection
      2. Unit 10.14 Transfection Using DEAE-Dextran
      3. Unit 10.15 Transfection by Electroporation
      4. Unit 10.16 Liposome-Mediated Transfection
      5. Unit 10.17A Stable Transfer of Genes into Mammalian Cells
      6. Unit 10.17B Transfection of Lymphoid Cells
      7. Unit 10.17C Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell–Based Systems
      8. Unit 10.17D Use of the Hemagglutinating Virus of Japan (HVJ) Envelope as a Versatile Delivery System for Nucleic Acids and Proteins to Leukocytes In Vitro
      9. Unit 10.17E Efficient In Vivo Delivery of Plasmids and Oligonucleotides Using Hemagglutinating Virus of Japan Envelope (HVJ-E) Vector in Immunological Disease Models
      10. Unit 10.18 Use of Monoclonal Antibodies for Expression Cloning
      11. Unit 10.19A Construction of Immunoglobulin Fusion Proteins
      12. Unit 10.19B Phage Display of Single-Chain Antibody Constructs
    8. Section VII The Polymerase Chain Reaction
      1. Unit 10.20 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
      2. Unit 10.21 Quantitation of Rare DNAs by PCR
      3. Unit 10.22 Detection of Isotype Switch Rearrangement in Bulk Culture by PCR
      4. Unit 10.23 Detection of Cytokine mRNA Expression by PCR
      5. Unit 10.24 cDNA Amplification Using One-Sided (Anchored) PCR
      6. Unit 10.25 Double-Stranded DNA Sequence Analysis
      7. Unit 10.26 Evaluating Human T Cell Receptor Gene Expression by PCR
      8. Unit 10.27 Molecular Analysis of Mouse T Cell Receptor Expression Using PCR
      9. Unit 10.28 Spectratype/Immunoscope Analysis of the Expressed TCR Repertoire
      10. Unit 10.29 Multiprobe Ribonuclease Protection Assay for Simultaneous Measurement of mRNA Expression
      11. Unit 10.30 Analysis of Telomere Length and Telomerase Activity
      12. Unit 10.31 Measurement of Mouse T Cell Receptor Excision Circles
      13. Unit 10.32 Use of Bacterial Artificial Chromosomes in Generating Targeted Mutations in Human and Mouse Cytomegaloviruses
      14. Unit 10.33 Unbiased Molecular Analysis of T Cell Receptor Expression Using Template-Switch Anchored RT-PCR
  13. Chapter 11 Biochemistry of Cell Activation
    1. Introduction
    2. Unit 11.1 Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation
    3. Unit 11.2 Preparation and Analysis of Phosphorylated Proteins
    4. Unit 11.3 Detecting Tyrosine-Phosphorylated Proteins by Western Blot Analysis
    5. Unit 11.4 Immune-Complex Assays for Tyrosine Protein Kinases
    6. Unit 11.5 Assessment of the Effects of Tyrosine Protein Kinase Inhibitors
    7. Unit 11.6 Production of Antibodies that Recognize Specific Tyrosine-Phosphorylated Peptides
    8. Unit 11.7 Protein Tyrosine Phosphatase Assays
    9. Unit 11.8 Analyzing Mitogen-Activated Protein Kinase (MAPK) Activities in T Cells
    10. Unit 11.9A Key Molecules Involved in Receptor-Mediated Lymphocyte Activation
    11. Unit 11.9B Signal Transduction During Activation and Inhibition of Natural Killer Cells
    12. Unit 11.9C Apoptosis Signaling Pathways
    13. Unit 11.9D TRAF-Mediated TNFR-Family Signaling
    14. Unit 11.10 Isolation and Use of Rafts
  14. Chapter 12 Detection and Analysis of HIV
    1. Introduction
    2. Unit 12.1 General Guidelines for Experimenting with HIV
    3. Unit 12.2 Isolation and Quantitation of HIV in Peripheral Blood
    4. Unit 12.3 Infection of CD4+ Primary T Cells and Cell Lines, Generation of Chronically Infected Cell Lines, and Induction of HIV Expression
    5. Unit 12.4 Culture of HIV in Monocytes and Macrophages
    6. Unit 12.5 Detection Assays for HIV Proteins
    7. Unit 12.6 Detection of HIV DNA and RNA Using PCR
    8. Unit 12.7 Regulation of the HIV Promoter/Enhancer
    9. Unit 12.8 In Situ Hybridization for Detection of HIV RNA
    10. Unit 12.9 In Vitro Evaluation of Experimental Agents for Anti-HIV Activity
    11. Unit 12.10 Vaccinia-Based Reporter Gene Cell-Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors
    12. Unit 12.11 Evaluating Neutralizing Antibodies Against HIV, SIV, and SHIV in Luciferase Reporter Gene Assays
    13. Unit 12.12 Immunophenotyping of T Cell Subpopulations in HIV Disease
  15. Chapter 13 Complement
    1. Introduction
    2. Unit 13.1 Classical Pathway Evaluation
    3. Unit 13.2 Alternative Pathway Evaluation
    4. Unit 13.3 Isolation and Purification of C3 from Human Plasma
    5. Unit 13.4 Assays for Membrane Complement Receptors
    6. Unit 13.5 Isolation and Functional Assay of the Membrane Complement Inhibitors CD55 (DAF) and CD59 (MIRL)
    7. Unit 13.6 Assays for the Mannan-Binding Lectin Pathway
    8. Unit 13.7 Assays for Complement Proteins Encoded in the Class III Region of Human MHC
    9. Unit 13.8 Human Complement Components C4A and C4B Genetic Diversities: Complex Genotypes and Phenotypes
  16. Chapter 14 Innate Immunity
    1. Introduction
    2. Unit 14.1 The Isolation and Characterization of Murine Macrophages
    3. Unit 14.2 Activation of Murine Macrophages
    4. Unit 14.3 Characterization of Human Monocytes/Macrophages
    5. Unit 14.4 Activation of Murine Macrophages
    6. Unit 14.5 Oxidative Metabolism of Murine Macrophages
    7. Unit 14.6 Measurement of Bacterial Ingestion and Killing by Macrophages
    8. Unit 14.7 Assay for Macrophage-Mediated Anti-Tumor Cytotoxicity
    9. Unit 14.8 Measurement of Fcgamma Receptor–Mediated Binding and Phagocytosis
    10. Unit 14.9 Measurement of Interferon-Mediated Antiviral Activity of Macrophages
    11. Unit 14.10 Human Natural Killer Receptors, Co-Receptors, and Their Ligands
    12. Unit 14.11 Isolation and Functional Use of Human NKT Cells
    13. Unit 14.12 Toll-Like Receptors
    14. Unit 14.13 Characterization and Functional Analysis of Mouse Invariant Natural T (iNKT) Cells
    15. Unit 14.14 Measuring Autophagy in Macrophages
    16. Unit 14.15 Monocyte and Neutrophil Isolation and Migration Assays
    17. Unit 14.16 Measuring TLR Function in Transfectants
    18. Unit 14.17 Measurement of Myeloid Cell Immune Suppressive Activity
    19. Unit 14.18 Measurement of Tumor Cytolysis by Macrophages
    20. Unit 14.19 In Vivo and Ex Vivo Protocols for Measuring the Killing of Extracellular Pathogens by Macrophages
    21. Unit 14.20 Measurement of Macrophage Growth and Differentiation
    22. Unit 14.21 Measurement of Type I Interferon Production
    23. Unit 14.22 Quantitative Assessment of Macrophage Functions in Repair and Fibrosis
    24. Unit 14.23 Measuring the Killing of Intracellular Pathogens: Leishmania
    25. Unit 14.24 Measuring Matrix Metalloproteinase Activity in Macrophages and Polymorphonuclear Leukocytes
    26. Unit 14.25 Measurement of Macrophage-Mediated Killing of Intracellular Bacteria, Including Francisella and Mycobacteria
    27. Unit 14.26 Metabolomics-Lipidomics of Eicosanoids and Docosanoids Generated by Phagocytes
    28. Unit 14.27 Measuring Opsonic Phagocytosis via Fcgamma Receptors and Complement Receptors on Macrophages
    29. Unit 14.28 Analysis of Gene Expression and Gene Silencing in Human Macrophages
    30. Unit 14.29 Target-Induced Natural Killer Cell Loss as a Measure of NK Cell Responses
  17. Chapter 15 Animal Models for Autoimmune and Inflammatory Disease
    1. Introduction
    2. Unit 15.1 Experimental Autoimmune Encephalomyelitis in the Mouse
    3. Unit 15.2 Experimental Autoimmune Encephalomyelitis in the Rat
    4. Unit 15.3 The BB Rat as a Model of Human Insulin-Dependent Diabetes Mellitus
    5. Unit 15.4 Adjuvant Arthritis in the Rat
    6. Unit 15.5 Collagen-Induced Arthritis
    7. Unit 15.6 Experimental Autoimmune Uveoretinitis in the Rat and Mouse
    8. Unit 15.7 Experimental Autoimmune Thyroiditis in the Mouse
    9. Unit 15.8 Experimental Autoimmune Myasthenia Gravis in the Mouse
    10. Unit 15.9 The NOD Mouse: A Model for Insulin-Dependent Diabetes Mellitus
    11. Unit 15.10 Streptococcal Cell Wall Arthritis
    12. Unit 15.11 Animal Models of IgA Nephropathy
    13. Unit 15.12 Induction of Autoimmune Disease by Depletion of Regulatory T Cells
    14. Unit 15.13 Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Depletion of Regulatory T Cells
    15. Unit 15.14 Autoimmune Myocarditis
    16. Unit 15.15 Mercuric Chloride–Induced Autoimmunity
    17. Unit 15.16 The Day-3 Thymectomy Model for Induction of Multiple Organ-Specific Autoimmune Diseases
    18. Unit 15.17 Autoimmune Ovarian Disease Induced by Immunization with Zona Pellucida (ZP3) Peptide
    19. Unit 15.18 Animal Models of Airway Sensitization
    20. Unit 15.19 Induction of TNBS Colitis in Mice
    21. Unit 15.20 Animal Models for SLE
    22. Unit 15.21 Creation of “Humanized” Mice to Study Human Immunity
    23. Unit 15.22 The K/BxN Arthritis Model
    24. Unit 15.23 Experimental Autoimmune Myasthenia Gravis in the Mouse
    25. Unit 15.24 Mouse Models of Atherosclerosis
  18. Chapter 16 Antigen Processing and Presentation
    1. Introduction
    2. Unit 16.1 Choosing and Preparing Antigen-Presenting Cells
    3. Unit 16.2 Presenting Exogenous Antigen to T Cells
    4. Unit 16.3 Targeted Identification of Infection-Related HLA Class I–Presented Epitopes by Stable Isotope Tagging of Epitopes (SITE)
  19. Chapter 17 Engineering Immune Molecules and Receptors
    1. Introduction
    2. Unit 17.1 Bacteriophage Library Construction and Selection of Recombinant Antibodies
    3. Unit 17.2 Monitoring Antigen-Specific T Cells Using MHC-Ig Dimers
    4. Unit 17.3 MHC-Peptide Tetramers to Visualize Antigen-Specific T Cells
    5. Unit 17.4 In Situ Staining Using MHC Class I Tetramers
    6. Unit 17.5 Preparation of Stable Single-Chain Trimers Engineered with Peptide, beta2 Microglobulin, and MHC Heavy Chain
  20. Chapter 18 Ligand-Receptor Interactions in the Immune System
    1. Introduction
    2. Unit 18.1 Binding of Biotinylated Peptides to MHC Class II Proteins on Cell Surfaces
    3. Unit 18.2 Detection of Low-Affinity Ligand-Receptor Interactions at the Cell Surface with Fluorescent Microspheres
    4. Unit 18.3 Measurement of MHC/Peptide Interactions by Gel Filtration
    5. Unit 18.4 Measurement of Peptide-MHC Interactions in Solution Using the Spin Column Filtration Assay
    6. Unit 18.5 Measurement of the Kinetics of Biomolecular Interactions Using the IAsys Resonant Mirror Biosensor
    7. Unit 18.6 Affinity-Based Biosensors for Biomolecular Interaction Analysis
    8. Unit 18.7 Biotin Labeling and Quantitation of Cell-Surface Proteins
    9. Unit 18.8 Measuring Protein-Protein Interactions by Equilibrium Sedimentation
    10. Unit 18.9 Binding of Small Peptides to Immobilized Antibodies: Kinetic Analysis by Surface Plasmon Resonance
    11. Unit 18.10 Fluorescence Resonance Energy Transfer: Techniques for Measuring Molecular Conformation and Molecular Proximity
    12. Unit 18.11 Peptide Induction of Surface Expression of Class I MHC
    13. Unit 18.12 Competition-Based Cellular Peptide Binding Assay for HLA Class I
    14. Unit 18.13 Supported Planar Bilayers for Study of the Immunological Synapse
    15. Unit 18.14 Measurement of Peptide Dissociation from MHC Class II Molecules
    16. Unit 18.15 Characterizing Protein-Protein Interactions by Sedimentation Velocity Analytical Ultracentrifugation
    17. Unit 18.16 Generation of Peptide MHC Class I Monomers and Multimers Through Ligand Exchange
    18. Unit 18.17 The MHC Motif Viewer: A Visualization Tool for MHC Binding Motifs
    19. Unit 18.18 Analysis of the Targeting Properties of Fc Chimeric Proteins and Antibodies in Mice In Vivo
  21. Chapter 19 Animal Models for Infectious Diseases
    1. Introduction
    2. Unit 19.1 Schistosomiasis
    3. Unit 19.2 Animal Models for the Analysis of Immune Responses to Leishmaniasis
    4. Unit 19.3 Animal Models for Toxoplasma gondii Infection
    5. Unit 19.4 Experimental Asexual Blood Stage Malaria Immunity
    6. Unit 19.5 Animal Models of Mycobacteria Infection
    7. Unit 19.6 Animal Models for Candidiasis
    8. Unit 19.7 A Mouse Model for Cytomegalovirus Infection
    9. Unit 19.8 Helicobacter Animal Models
    10. Unit 19.9 Animal Model for Infection with Listeria monocytogenes
    11. Unit 19.10 Animal Models Using Lymphocytic Choriomeningitis Virus
    12. Unit 19.11 Influenza Virus
    13. Unit 19.12 Animal Model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus
    14. Unit 19.13 Cecal Ligation and Puncture
    15. Unit 19.14 Infection of Mice with Francisella as an Immunological Model
  22. Chapter 20 Animal Models for Tumor Immunology
    1. Introduction
    2. Unit 20.1 B16 as a Mouse Model for Human Melanoma
    3. Unit 20.2 Mouse 4T1 Breast Tumor Model
    4. Unit 20.3 Mouse SaI Sarcoma Tumor Model
    5. Unit 20.4 The P815 Mastocytoma Tumor Model
    6. Unit 20.5 The TRAMP Mouse as a Model for Prostate Cancer
    7. Unit 20.6 Models for Lymphoma
    8. Unit 20.7 Identification of Human Tumor Antigens by Serological Analysis of Recombinant cDNA Expression Libraries (SEREX)
    9. Unit 20.8 Carcinoembryonic Antigen Transgenic Mouse Models for Immunotherapy and Development of Cancer Vaccines
    10. Unit 20.9 ErbB2 Transgenic Mice: A Tool for Investigation of the Immune Prevention and Treatment of Mammary Carcinomas
    11. Unit 20.10 Molecular Cloning and Characterization of MHC Class I– and II–Restricted Tumor Antigens Recognized by T Cells
    12. Unit 20.11 The Immune Response to Tumors
  23. Chapter 21 Microscopy
    1. Introduction
    2. Unit 21.1 Proper Alignment and Adjustment of the Light Microscope
    3. Unit 21.2 Fluorescence Microscopy
    4. Unit 21.3 Immunofluorescence Staining
    5. Unit 21.4 Immunohistochemistry
  24. Chapter 22 Isolation and Differentiation of Stem and Progenitor Cells
    1. Introduction
    2. Section A Sources and Methods to Obtain Stem and Progenitor Cells
      1. Unit 22A.1 Large Animal Models for Stem and Progenitor Cell Analysis
      2. Unit 22A.2 Sources of Human and Murine Hematopoietic Stem Cells
    3. Section B Isolation of Murine Stem and Progenitor Cells
      1. Unit 22B.1 Isolation of Murine Hematopoietic Stem Cells and Progenitor Cells
      2. Unit 22B.2 Characterization of Mouse Hematopoietic Stem and Progenitor Cells
    4. Section F Differentiation of Special Populations of Stem and Progenitor Cells
      1. Unit 22F.1 Isolation of Prolymphocytes from Bone Marrow and Fetal Liver
      2. Unit 22F.2 In Vitro Differentiation and Measurement of B Cell Progenitor Activity in Culture
      3. Unit 22F.3 Measurement of Natural Killer Cell Progenitor Activity in Culture
      4. Unit 22F.4 Differentiation of Peripheral Blood Monocytes into Dendritic Cells
      5. Unit 22F.5 Differentiation and Characterization of Myeloid Cells
      6. Unit 22F.6 Culture, Expansion, and Differentiation of Murine Megakaryocytes
      7. Unit 22F.7 Differentiation of Human Erythroid Cells in Culture
      8. Unit 22F.8 Differentiation and Characterization of T Cells
      9. Unit 22F.9 Differentiation of Multipotent Adult Progenitor Cells into Functional Endothelial and Smooth Muscle Cells
      10. Unit 22F.10 Differentiation of Mast Cells from Embryonic Stem Cells
  25. Appendix 1 Abbreviations and Useful Data
    1. 1A Abbreviations Used in this Manual
    2. 1B Amino Acids
    3. 1C Commonly Used Mouse Strains
    4. 1D Commonly Used Detergents
    5. 1E Murine Models of Immunodeficiency and Autoimmune Disease
    6. 1F Mouse Endogenous Superantigens: Ms and Mls-like Determinants Encoded by Mouse Retroviruses
    7. 1G HLA Type of EBV-Transformed Human B Cell Lines
    8. 1H Commonly Used Rat Strains
    9. 1I Peptide Binding Motifs for MHC Class I and II Molecules
    10. 1J Web Sites of Interest to Immunologists
    11. 1K Human MHC Genes and Products
    12. 1L Mouse MHC Genes and Products
    13. 1M Listing, Location, Binding Motifs, and Expression of Nonclassical Class I and Related Genes and Molecules
    14. 1N Overview of Protein Folds in the Immune System
    15. 1O Nomenclature of the Human T Cell Receptor Genes
    16. 1P Nomenclature of the Human Immunoglobulin Genes
    17. 1Q Safe Use of Radioisotopes
    18. 1R Receptors Involved in the Regulation of Murine NK Cells
    19. 1S Nomenclature and Serology of HLA Class I and Class II Alleles
    20. 1T Heat-Shock Proteins
    21. 1U Large-Scale Cell Culture
    22. 1V Biosafety: Guidelines for Working with Pathogenic and Infectious Microorganisms
    23. 1W Using Bioinformatics Tools for the Sequence Analysis of Immunoglobulins and T Cell Receptors
    24. 1X Paired Receptor Systems of the Innate Immune System
  26. Appendix 2 Commonly Used Reagents and Equipment
    1. 2A Common Media, Buffers, and Stock Solutions
    2. 2B Equipment
  27. Appendix 3 Commonly Used Immunological Techniques
    1. 3A Monitoring Cell Growth
    2. 3B Trypan Blue Exclusion Test of Cell Viability
    3. 3C Wright-Giemsa and Nonspecific Esterase Staining of Cells
    4. 3D Labeling Cells in Microtiter Plates for Determination of [3H]Thymidine Uptake
    5. 3E Diagnosis and Treatment of Mycoplasma-Contaminated Cell Cultures
    6. 3F Obtaining Human Peripheral Blood Cells
    7. 3G Cryopreservation of Cells
    8. 3H Dialysis and Concentration of Protein Solutions
    9. 3I Column Chromatography of Antibodies and Fragments
    10. 3J Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots
    11. 3K Silanizing Glassware
    12. 3L Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
    13. 3M Preparing Frozen Bacterial Stocks
    14. 3N Transformation of E. coli by Electroporation
    15. 3O Gram Staining
  28. Appendix 4 The CD System of Leukocyte Surface Molecules
    1. 4A Monoclonal Antibodies to Human Cell Surface Antigens
    2. 4B Monoclonal Antibodies to Mouse Cell-Surface Antigens
    3. 4C Monoclonal Antibodies to Rat Leukocyte Surface Antigens, MHC Antigens, and Immunoglobulins
  29. Appendix 5 Suppliers
    1. 5 Selected Suppliers of Reagents and Equipment

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