Current Protocols in Immunology

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Current Protocols in Immunology is a "best-practices" collection that provides comprehensive coverage of immunological methods from classic to the most cutting edge: the most sophisticated protocols in immunology today.

Updated every three months in all formats, CPI is constantly evolving to keep pace with the very latest discoveries and developments. A year of these updates is included in the initial CPI purchase price. That's 770 pages of new (43%) and revised (57%) content on average every year since the initial publication of the work in January 1991! Presently five volumes in its looseleaf print version, CPI...

* covers the latest in antibody detection and preparation, molecular immunology, innate immunity, tumor immunology, engineering immune molecules and receptors, and more.
* includes a recently-added chapter on sources and proven methods for isolation and characterization of stem and progenitor cells, as well as animal models for analyses.
* offers valuable reference information in five extensive appendices, including commonly used reagents, equipment and techniques; and a full appendix devoted to the CD system of leukocyte surface molecules.

Edited by: John E. Coligan (NIAID, NIH); Barbara Bierer (Brigham and Women's Hospital and Harvard Medical School); David H. Margulies (NIAID, NIH); Ethan M. Shevach (NIAID, NIH); Warren Strober (NIAID, NIH); Richard Coico (Weill Medical College of Cornell University); Guest Editors: Patricia Brown (Frederick, Maryland); John C. Donovan (Wyeth Research); Past Editor: Ada Kruisbeek (Netherlands Research Institute)


Chapter 1 Care and Handling of Laboratory Animals
Introduction
Section I Animal Health and Husbandry
Unit 1.1 Animal Health Assurance
Unit 1.2 Managing Immunocompromised Animals
Unit 1.3 Handling and Restraint
Unit 1.4 Anesthesia
Unit 1.5 Animal Identification
Section II Use of Mouse, Rat, Hamster, and Rabbit
Unit 1.6 Parenteral Injections
Unit 1.7 Blood Collection
Unit 1.8 Euthanasia
Section III Selected Surgical Procedures
Unit 1.9 Removal of Lymphoid Organs
Unit 1.10 Survival Surgery: Removal of the Spleen or Thymus
Unit 1.11 Surgical Removal of Mouse Salivary Glands

Chapter 2 Induction of Immune Responses
Introduction
Section I Assays for Antibody Production
Unit 2.1 Enzyme-Linked Immunosorbent Assays
Unit 2.2 Isotype Determination of Antibodies
Unit 2.3 Double-Immunodiffusion Assay for Detecting Specific Antibodies
Section II Production of Antibodies
Unit 2.4 Production of Polyclonal Antisera
Unit 2.5 Production of Monoclonal Antibodies
Unit 2.6 Monoclonal Antibody Supernatant and Ascites Fluid Production
Section III Purification and Fragmentation of Antibodies
Unit 2.7 Purification of Immunoglobulin G
Unit 2.8 Fragmentation of Immunoglobulin G
Unit 2.9 Purification of Immunoglobulin M and Immunoglobulin D
Unit 2.10A Fragmentation of Immunoglobulin M
Unit 2.10B Purification of Human IgA
Section IV Specialized Techniques
Unit 2.11 Preparation of Immune Stimulating Complexes (ISCOMs) as Adjuvants
Unit 2.12 Cloning, Expression, and Modification of Antibody V Regions
Unit 2.13 Production of Bispecific Antibodies
Unit 2.14 Nucleic Acid Immunizations

Chapter 3 In Vitro Assays for Mouse Lymphocyte Function
Introduction
Section I Isolation and Fractionation of Mononuclear Cell Populations
Unit 3.1 Isolation of Mouse Mononuclear Cells
Unit 3.2 T Cell Enrichment By Nonadherence to Nylon
Unit 3.3 T Cell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells
Unit 3.4 T Cell Depletion by Cytotoxic Elimination
Unit 3.5A Fractionation of T and B Cells Using Magnetic Beads
Unit 3.5B Isolation of Ly-1+/CD5+ B Cells by Cell Sorting
Unit 3.6 Depletion of Accessory Cells by Adherence to Sephadex G-10
Unit 3.7 Isolation of Dendritic Cells
Section II Assays for B Cell Function
Unit 3.8 Assays for B Lymphocyte Function
Unit 3.9 Early Events in B Lymphocyte Activation
Unit 3.10 Proliferative Assays for B Cell Function
Section III Assays for T Cell Function
Unit 3.11 Induction and Measurement of Cytotoxic T Lymphocyte Activity
Unit 3.12 Proliferative Assays for T Cell Function
Unit 3.13 Production of TH1 and TH2 Cell Lines and Clones
Unit 3.14 Production of Mouse T Cell Hybridomas
Unit 3.15 Quantitation of Functional T Cells by Limiting Dilution
Unit 3.16 Granule Enzyme Exocytosis Assay for Cytotoxic T Lymphocyte Activation
Section IV Related Isolation Procedures and Functional Assays
Unit 3.17 Measurement of Apoptosis and Other Forms of Cell Death
Unit 3.18 In Vitro Systems for the Study of T Cell Development: Fetal Thymus Organ Culture and OP9-DL1 Cell Coculture
Unit 3.19 Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes, Peyer's Patch, and Lamina Propria Cells
Unit 3.20 Isolation of Mouse Neutrophils
Unit 3.21 Isolation of Mouse Intrahepatic Lymphocytes
Unit 3.22 Isolation of Murine Natural Killer Cells
Unit 3.23 Generation, Isolation, and Maintenance of Rodent Mast Cells and Mast Cell Lines

Chapter 4 In Vivo Assays for Lymphocyte Function
Introduction
Unit 4.1 In Vivo Depletion of CD4- and CD8-Specific T Cells
Unit 4.2 Contact Hypersensitivity
Unit 4.3 Animal Models of Acute and Chronic Graft-Versus-Host Disease
Unit 4.4 Skin Allograft Rejection
Unit 4.5 Delayed-Type Hypersensitivity
Unit 4.6 Assessment of Lymphocyte Development in Radiation Bone Marrow Chimeras
Unit 4.7 Measurement of T and B Cell Turnover with Bromodeoxyuridine
Unit 4.8 Construction of Human-SCID Chimeric Mice
Unit 4.9 Use of the Intracellular Fluorescent Dye CFSE to Monitor Lymphocyte Migration and Proliferation

Chapter 5 Immunofluorescence and Cell Sorting
Introduction
Unit 5.1 Overview of Flow Cytometry
Unit 5.2 Analysis of Flow Cytometry Data
Unit 5.3 Preparation of Cells and Reagents for Flow Cytometry
Unit 5.4 Flow Cytometry Analysis Using the Becton Dickinson FACS Calibur
Unit 5.5 Measurement of Intracellular Ions by Flow Cytometry
Unit 5.6 Measurement of Intercellular Conjugates by Flow Cytometry
Unit 5.7 Analysis of Cellular DNA Content by Flow Cytometry
Unit 5.8 Multiparameter FACS Analysis

Chapter 6 Cytokines and Their Cellular Receptors
Introduction
Unit 6.1 Measurement of Lymphokine Receptors
Unit 6.2 Measurement of Soluble and Membrane-Bound Interleukin 1 Using a Fibroblast Bioassay
Unit 6.3 Measurement of Human and Murine Interleukin 2 and Interleukin 4
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
Unit 6.5 Measurement of Mouse and Human Interleukin 5
Unit 6.6 Measurement of Interleukin 6
Unit 6.7 Measurement of Interleukin 7
Unit 6.8 Measurement of Mouse and Human Interferon
Unit 6.9 Measurement of Antiviral Activity Induced by Interferons , , and
Unit 6.10 Measurement of Tumor Necrosis Factor and
Unit 6.11 Measurement of TGF- in Biological Fluids
Unit 6.12 Biological Responses to Chemokine Superfamily Members
Unit 6.13 Measurement of Mouse and Human Interleukin 9
Unit 6.14 Measurement of Mouse and Human Interleukin 10
Unit 6.15 Measurement of Human Interleukin 11
Unit 6.16 Measurement of Human and Mouse Interleukin-12
Unit 6.17 Measurement of Human and Murine Stem Cell Factor (c-kit Ligand)
Unit 6.18 Measurement of Interleukin-13
Unit 6.19 ELISPOT Assay to Detect Cytokine-Secreting Murine and Human Cells
Unit 6.20 Immunoenzymetric Assay of Mouse and Human Cytokines Using NIP-Labeled Anti-Cytokine Antibodies
Unit 6.21 Detection of Cytokine Receptors by Flow Cytometry
Unit 6.22 Measurement of Interleukin 15
Unit 6.23 Measurement of Interleukin 16
Unit 6.24 Detection of Intracellular Cytokines by Flow Cytometry
Unit 6.25 Measurement of Interleukin 17
Unit 6.26 Measurement of Human and Mouse Interleukin 18
Unit 6.27 Detection and Isolation of Cytokine Secreting Cells Using the Cytometric Cytokine Secretion Assay
Unit 6.28 The In Vivo Cytokine Capture Assay for Measurement of Cytokine Production in the Mouse
Unit 6.29 Cytokine, Growth Factor, and Chemokine Ligand Database

Chapter 7 Immunologic Studies in Humans
Introduction
Section I Preparation of Human Mononuclear Cell Populations and Subpopulations
Unit 7.1 Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood
Unit 7.2 Isolation of T Cells Using Rosetting Procedures
Unit 7.3 Purification of T Cell Subpopulations
Unit 7.4 Immunomagnetic Purification of T Cell Subpopulations
Unit 7.4B The Purification and Functional Analysis of Human CD4+CD25high Regulatory T Cells
Unit 7.5 Isolation of B Cell Populations
Unit 7.6A Isolation of Human Monocyte Populations
Unit 7.6B Isolation and Purification of Human Intestinal Macrophages
Unit 7.7 Isolation of Human NK Cells and Generation of LAK Activity
Unit 7.8 Isolation of Mononuclear Cells From Tonsillar Tissue
Unit 7.9 Detection of Unseparated Human Lymphocytes by Flow Cytometry
Section II Measurement of Basic Immunologic Characteristics of Human Mononuclear Cells
Unit 7.10 Measurement of Proliferative Responses of Cultured Lymphocytes
Unit 7.11 Induction of Polyclonal Immunoglobulin Synthesis
Unit 7.12 Measurement of Polyclonal Immunoglobulin Synthesis Using ELISA
Unit 7.13 Measurement of Polyclonal Immunoglobulin Synthesis Using the Reverse Plaque Technique
Unit 7.14 Measurement of Immunoglobulin Synthesis Using the ELISPOT Assay
Unit 7.15 Measurement of In Vitro Specific-Antibody Synthesis
Unit 7.16 Induction and Measurement of In Vivo Antibody Responses
Unit 7.17 Measurement of Polyclonal and Antigen-Specific Cytotoxic T Cell Function
Unit 7.18 Measurement of Cytotoxic Activity of NK/LAK Cells
Unit 7.18B Measurement of Cytokine Production Using Whole Blood
Section III Long-Term and Immortalized Human Cell Lines and Clones
Unit 7.19 Generation and Maintenance of Cloned Human T Cell Lines
Unit 7.20 Generation of HTLV-I-Transformed T Cell Lines
Unit 7.21 Herpesvirus saimiri Transformation of Human T Lymphocytes
Unit 7.22 Generation of Epstein-Barr Virus (EBV)—Immortalized B Cell Lines
Section IV Preparation and Functional Analysis of Other Human Lymphoid and Nonlymphoid Cells
Unit 7.23 Isolation and Functional Analysis of Neutrophils
Unit 7.24 Isolation of Human Basophils
Unit 7.25 Isolation of Tissue Mast Cells
Unit 7.26 Measuring Degranulation of Mast Cells
Unit 7.27 51Cr Release Assay of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
Unit 7.28 Measurement of Cellular Adhesion Under Static Conditions
Unit 7.29 Cell Adhesion Under Hydrodynamic Flow Conditions
Unit 7.30 Isolation of Human Intestinal Mucosal Mononuclear Cells
Unit 7.31 Isolation of Human Eosinophils
Unit 7.32 Isolation and Generation of Human Dendritic Cells
Unit 7.33 Measurement of Prostaglandins and Other Eicosanoids
Unit 7.34 Isolation and Characterization of Human Natural Killer Cell Subsets
Unit 7.35 Assays for B Cell and Germinal Center Development
Unit 7.36 In Vitro Models of Human T Cell Anergy

Chapter 8 Isolation and Analysis of Proteins
Introduction
Section I Solubilization of Cellular Proteins
Unit 8.1A Solubilization of Lymphocytes
Unit 8.1B Purification of Organelles from Mammalian Cells
Unit 8.1C Subcellular Fractionation of Tissue Culture Cells
Section II Isolation of Proteins Using Antibodies
Unit 8.2 Immunoaffinity Chromatography
Unit 8.3 Immunoprecipitation
Section III Electrophoretic Separation of Proteins
Unit 8.4 One-Dimensional Gel Electrophoresis of Proteins
Unit 8.5 Two-Dimensional Gel Electrophoresis
Unit 8.6 Diagonal Gel Electrophoresis
Unit 8.7 Isolation of Proteins for Microsequence Analysis
Unit 8.8 Electroelution of Proteins from Stained Gels
Section IV Detection of Proteins
Unit 8.9 Staining Proteins in Gels
Unit 8.10 Immunoblotting and Immunodetection
Unit 8.11 Iodination of Soluble and Membrane-Bound Proteins
Unit 8.12 Biosynthetic Labeling of Proteins
Unit 8.13 Metabolic Radiolabeling of Animal Cell Glycoconjugates
Unit 8.14 Inhibition of N-Linked Glycosylation
Unit 8.15 Release of Saccharides from Glycoconjugates
Unit 8.16 Biotinylation and Analysis of Membrane-Bound and Soluble Proteins

Chapter 9 Peptides
Introduction
Unit 9.1 Introduction to Peptide Synthesis
Unit 9.2 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins
Unit 9.3 Production of Anti-Peptide Antisera
Unit 9.4 B Cell Epitope Mapping Using Synthetic Peptides
Unit 9.5 Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries
Unit 9.6 Synthesis and Application of Peptide Dendrimers As Protein Mimetics
Unit 9.7 Synthesis of Multiple Peptides on Plastic Pins
Unit 9.8 Phage Display Selection and Analysis of Ab-Binding Epitopes

Chapter 10 Molecular Biology
Introduction
Section I Isolation and Purification of DNA
Unit 10.1 Purification and Concentration of DNA from Aqueous Solutions
Unit 10.2 Preparation of Genomic DNA from Mammalian Tissue
Unit 10.3 Preparation of Bacterial Plasmid DNA
Section II Resolution and Recovery of DNA Fragments
Unit 10.4 Agarose Gel Electrophoresis
Unit 10.5 Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels
Section III Analysis of Genomic DNA by Blotting and Hybridization
Unit 10.6A Southern Blotting
Unit 10.6B Hybridization Analysis of DNA Blots
Unit 10.7 Deprotection of Oligonucleotides and Purification Using Denaturing PAGE
Section IV Enzymatic Cleavage and Labeling of DNA
Unit 10.8 Digestion of DNA with Restriction Endonucleases
Unit 10.9 Reagents and Radioisotopes Used to Manipulate Nucleic Acids
Unit 10.10 Enzymatic Labeling of Nucleic Acids
Section V Preparation and Analysis of RNA
Unit 10.11 Preparation of RNA from Tissues and Cells
Unit 10.12 Analysis of RNA by Northern and Slot-Blot Hybridization
Section VI Transfection and Expression of Cloned DNA
Unit 10.13 Calcium Phosphate Transfection
Unit 10.14 Transfection Using DEAE-Dextran
Unit 10.15 Transfection by Electroporation
Unit 10.16 Liposome-Mediated Transfection
Unit 10.17A Stable Transfer of Genes into Mammalian Cells
Unit 10.17B Transfection of Lymphoid Cells
Unit 10.17C Rapid Production of Retroviruses for Efficient Gene Delivery to Mammalian Cells Using 293T Cell—Based Systems
Unit 10.18 Use of Monoclonal Antibodies for Expression Cloning
Unit 10.19A Construction of Immunoglobulin Fusion Proteins
Unit 10.19B Phage Display of Single-Chain Antibody Constructs
Section VII The Polymerase Chain Reaction
Unit 10.20 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Unit 10.21 Quantitation of Rare DNAs by PCR
Unit 10.22 Detection of Isotype Switch Rearrangement in Bulk Culture by PCR
Unit 10.23 Detection of Cytokine mRNA Expression by PCR
Unit 10.24 cDNA Amplification Using One-Sided (Anchored) PCR
Unit 10.25 Double-Stranded DNA Sequence Analysis
Unit 10.26 Evaluating Human T Cell Receptor Gene Expression by PCR
Unit 10.27 Molecular Analysis of Mouse T Cell Receptor Expression Using PCR
Unit 10.28 Spectratype/Immunoscope Analysis of the Expressed TCR Repertoire
Unit 10.29 Multiprobe Ribonuclease Protection Assay for Simultaneous Measurement of mRNA Expression
Unit 10.30 Analysis of Telomere Length and Telomerase Activity
Unit 10.31 Measurement of Mouse T Cell Receptor Excision Circles

Chapter 11 Biochemistry of Cell Activation
Introduction
Unit 11.1 Analysis of Inositol Phospholipid Turnover During Lymphocyte Activation
Unit 11.2 Preparation and Analysis of Phosphorylated Proteins
Unit 11.3 Antiphosphotyrosine Blotting
Unit 11.4 Immune-Complex Assays for Tyrosine Protein Kinases
Unit 11.5 Assessment of the Effects of Tyrosine Protein Kinase Inhibitors
Unit 11.6 Production of Antibodies that Recognize Specific Tyrosine-Phosphorylated Peptides
Unit 11.7 Using Protein Kinase and Protein Phosphatase Inhibitors to Dissect Protein Phosphorylation Pathways
Unit 11.8 Analyzing Mitogen-Activated Protein Kinase (MAPK) Activities in T Cells
Unit 11.9A Key Molecules Involved in Receptor-Mediated Lymphocyte Activation
Unit 11.9B Signal Transduction During Natural Killer Cell Activation
Unit 11.9C Apoptosis Signaling Pathways
Unit 11.9D TRAF-Mediated TNFR-Family Signaling
Unit 11.10 Isolation and Use of Rafts

Chapter 12 Detection and Analysis of HIV
Introduction
Unit 12.1 General Guidelines for Experimenting with HIV
Unit 12.2 Isolation and Quantitation of HIV in Peripheral Blood
Unit 12.3 Infection of CD4+ Primary T Cells and Cell Lines, Generation of Chronically Infected Cell Lines, and Induction of HIV Expression
Unit 12.4 Culture of HIV in Monocytes and Macrophages
Unit 12.5 Detection Assays for HIV Proteins
Unit 12.6 Detection of HIV DNA and RNA Using PCR
Unit 12.7 Regulation of the HIV Promoter/Enhancer
Unit 12.8 In Situ Hybridization for Detection of HIV RNA
Unit 12.9 In Vitro Evaluation of Experimental Agents for Anti-HIV Activity
Unit 12.10 Vaccinia-Based Reporter Gene Cell-Fusion Assays to Quantitate Functional Interactions of HIV Envelope Glycoprotein with Receptors
Unit 12.11 Evaluating Neutralizing Antibodies Against HIV, SIV, and SHIV in Luciferase Reporter Gene Assays
Unit 12.12 Immunophenotyping of T Cell Subpopulations in HIV Disease

Chapter 13 Complement
Introduction
Unit 13.1 Classical Pathway Evaluation
Unit 13.2 Alternative Pathway Evaluation
Unit 13.3 Isolation and Purification of C3 from Human Plasma
Unit 13.4 Assays for Membrane Complement Receptors
Unit 13.5 Isolation and Functional Assay of the Membrane Complement Inhibitors CD55 (DAF) and CD59 (MIRL)
Unit 13.6 Assays for the Mannan-Binding Lectin Pathway
Unit 13.7 Assays for Complement Proteins Encoded in the Class III Region of Human MHC
Unit 13.8 Human Complement Components C4A and C4B Genetic Diversities: Complex Genotypes and Phenotypes

Chapter 14 Innate Immunity
Introduction
Unit 14.1 Isolation of Murine Macrophages
Unit 14.2 Characterization of Murine Macrophages
Unit 14.3 Characterization of Human Monocytes/Macrophages
Unit 14.4 Activation of Murine Macrophages
Unit 14.5 Oxidative Metabolism of Murine Macrophages
Unit 14.6 Measurement of Bacterial Ingestion and Killing by Macrophages
Unit 14.7 Assay for Macrophage-Mediated Anti-Tumor Cytotoxicity
Unit 14.8 Measurement of Fc Receptor—Mediated Binding and Phagocytosis
Unit 14.9 Measurement of Interferon-Mediated Antiviral Activity of Macrophages
Unit 14.10 Human Natural Killer Receptors and Their Ligands
Unit 14.11 Isolation and Functional Use of Human NK T Cells
Unit 14.12 Toll Receptors
Unit 14.13 Characterization and Functional Analysis of Mouse Invariant Natural T (iNKT) Cells

Chapter 15 Animal Models for Autoimmune and Inflammatory Disease
Introduction
Unit 15.1 Experimental Autoimmune Encephalomyelitis in the Mouse
Unit 15.2 Experimental Autoimmune Encephalomyelitis in the Rat
Unit 15.3 The BB Rat as a Model of Human Insulin-Dependent Diabetes Mellitus
Unit 15.4 Adjuvant Arthritis in the Rat
Unit 15.5 Collagen-Induced Arthritis
Unit 15.6 Experimental Autoimmune Uveoretinitis in the Rat and Mouse
Unit 15.7 Experimental Autoimmune Thyroiditis in the Mouse
Unit 15.8 Experimental Autoimmune Myasthenia Gravis in the Mouse
Unit 15.9 The NOD Mouse: A Model for Insulin-Dependent Diabetes Mellitus
Unit 15.10 Streptococcal Cell Wall Arthritis
Unit 15.11 Animal Models of IgA Nephropathy
Unit 15.12 Induction of Autoimmune Disease by Depletion of Regulatory T Cells
Unit 15.13 Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Depletion of Regulatory T Cells
Unit 15.14 Autoimmune Myocarditis
Unit 15.15 Mercuric Chloride—Induced Autoimmunity
Unit 15.16 The Day-3 Thymectomy Model for Induction of Multiple Organ-Specific Autoimmune Diseases
Unit 15.17 Autoimmune Ovarian Disease Induced by Immunization with Zona Pellucida (ZP3) Peptide
Unit 15.18 Animal Models of Airway Sensitization
Unit 15.19 Induction of TNBS Colitis in Mice
Unit 15.20 Animal Models for SLE

Chapter 16 Antigen Processing and Presentation
Introduction
Unit 16.1 Choosing and Preparing Antigen-Presenting Cells
Unit 16.2 Presenting Exogenous Antigen to T Cells

Chapter 17 Engineering Immune Molecules and Receptors
Introduction
Unit 17.1 Bacteriophage Library Construction and Selection of Recombinant Antibodies
Unit 17.2 Monitoring Antigen-Specific T Cells Using MHC-Ig Dimers
Unit 17.3 MHC-Peptide Tetramers to Visualize Antigen-Specific T Cells
Unit 17.4 In Situ Staining Using MHC Class I Tetramers

Chapter 18 Ligand-Receptor Interactions in the Immune System
Introduction
Unit 18.1 Binding of Biotinylated Peptides to MHC Class II Proteins on Cell Surfaces
Unit 18.2 Detection of Low-Affinity Ligand-Receptor Interactions at the Cell Surface with Fluorescent Microspheres
Unit 18.3 Measurement of MHC/Peptide Interactions by Gel Filtration
Unit 18.4 Measurement of Peptide-MHC Interactions in Solution Using the Spin Column Filtration Assay
Unit 18.5 Measurement of the Kinetics of Biomolecular Interactions Using the IAsys Resonant Mirror Biosensor
Unit 18.6 Affinity-Based Biosensors for Biomolecular Interaction Analysis
Unit 18.7 Biotin Labeling and Quantitation of Cell-Surface Proteins
Unit 18.8 Measuring Protein-Protein Interactions by Equilibrium Sedimentation
Unit 18.9 Binding of Small Peptides to Immobilized Antibodies: Kinetic Analysis by Surface Plasmon Resonance
Unit 18.10 Fluorescence Resonance Energy Transfer: Techniques for Measuring Molecular Conformation and Molecular Proximity
Unit 18.11 Peptide Induction of Surface Expression of Class I MHC
Unit 18.12 Competition-Based Cellular Peptide Binding Assay for HLA Class I

Chapter 19 Animal Models for Infectious Diseases
Introduction
Unit 19.1 Schistosomiasis
Unit 19.2 Animal Models for the Analysis of Immune Responses to Leishmaniasis
Unit 19.3 Animal Models for Toxoplasma gondii Infection
Unit 19.4 Experimental Asexual Blood Stage Malaria Immunity
Unit 19.5 Animal Models of Mycobacteria Infection
Unit 19.6 Animal Models for Candidiasis
Unit 19.7 A Mouse Model for Cytomegalovirus Infection
Unit 19.8 Helicobacter Animal Models
Unit 19.9 Animal Model for Infection with Listeria monocytogenes
Unit 19.10 Animal Models Using Lymphocytic Choriomeningitis Virus
Unit 19.11 Influenza Virus
Unit 19.12 Animal Model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus

Chapter 20 Animal Models for Tumor Immunology
Introduction
Unit 20.1 B16 as a Mouse Model for Human Melanoma
Unit 20.2 Mouse 4T1 Breast Tumor Model
Unit 20.3 Mouse SaI Sarcoma Tumor Model
Unit 20.4 The P815 Mastocytoma Tumor Model
Unit 20.5 The TRAMP Mouse as a Model for Prostate Cancer
Unit 20.6 Models for Lymphoma
Unit 20.7 Identification of Human Tumor Antigens by Serological Analysis of Recombinant cDNA Expression Libraries (SEREX)

Chapter 21 Microscopy
Introduction
Unit 21.1 Proper Alignment and Adjustment of the Light Microscope
Unit 21.2 Fluorescence Microscopy
Unit 21.3 Immunofluorescence Staining
Unit 21.4 Immunohistochemistry

Chapter 22 Isolation and Differentiation of Stem and Progenitor Cells
Introduction
Section A Sources and Methods to Obtain Stem and Progenitor Cells
Unit 22A.1 Large Animal Models for Stem and Progenitor Cell Analysis
Section B Isolation of Murine Stem and Progenitor Cells
Unit 22B.1 Isolation of Murine Hematopoietic Stem Cells and Progenitor Cells
Section F Differentiation of Special Populations of Stem and Progenitor Cells
Unit 22F.1 Isolation of Prolymphocytes from Bone Marrow and Fetal Liver
Unit 22F.2 In Vitro Differentiation and Measurement of B Cell Progenitor Activity in Culture
Unit 22F.3 Measurement of Natural Killer Cell Progenitor Activity in Culture
Unit 22F.4 Differentiation of Peripheral Blood Monocytes into Dendritic Cells
Unit 22F.5 Differentiation and Characterization of Myeloid Cells
Unit 22F.6 Culture, Expansion, and Differentiation of Murine Megakaryocytes
Unit 22F.7 Differentiation of Human Erythroid Cells in Culture
Unit 22F.8 Differentiation and Characterization of T Cells

Appendix 1 Abbreviations and Useful Data
1A Abbreviations Used in this Manual
1B Amino Acids
1C Commonly Used Mouse Strains
1D Commonly Used Detergents
1E Murine Models of Immunodeficiency and Autoimmune Disease
1F Mouse Endogenous Superantigens: Ms and Mls-like Determinants Encoded by Mouse Retroviruses
1G HLA Type of EBV-Transformed Human B Cell Lines
1H Commonly Used Rat Strains
1I Peptide Binding Motifs for MHC Class I and II Molecules
1J Web Sites of Interest to Immunologists
1K Human MHC Genes and Products
1L Mouse MHC Genes and Products
1M Listing, Location, Binding Motifs, and Expression of Nonclassical Class I and Related Genes and Molecules
1N Overview of Protein Folds in the Immune System
1O Nomenclature of the Human T Cell Receptor Genes
1P Nomenclature of the Human Immunoglobulin Genes
1Q Safe Use of Radioisotopes
1R Receptors Involved in the Regulation of Murine NK Cells
1S Nomenclature and Serology of HLA Class I and Class II Alleles
1T Heat-Shock Proteins
1U Large-Scale Cell Culture
1V Biosafety: Guidelines for Working with Pathogenic and Infectious Microorganisms
1W Using Bioinformatics Tools for the Sequence Analysis of Immunoglobulins and T Cell Receptors

Appendix 2 Commonly Used Reagents and Equipment
2A Common Media, Buffers, and Stock Solutions
2B Equipment

Appendix 3 Commonly Used Immunological Techniques
3A Monitoring Cell Growth
3B Trypan Blue Exclusion Test of Cell Viability
3C Wright-Giemsa and Nonspecific Esterase Staining of Cells
3D Labeling Cells in Microtiter Plates for Determination of [3H]Thymidine Uptake
3E Diagnosis and Treatment of Mycoplasma-Contaminated Cell Cultures
3F Obtaining Human Peripheral Blood Cells
3G Cryopreservation of Cells
3H Dialysis and Concentration of Protein Solutions
3I Column Chromatography of Antibodies and Fragments
3J Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots
3K Silanizing Glassware
3L Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
3M Preparing Frozen Bacterial Stocks
3N Transformation of E. coli by Electroporation
3O Gram Staining

Appendix 4 The CD System of Leukocyte Surface Molecules
4A Monoclonal Antibodies to Human Cell Surface Antigens
4B Monoclonal Antibodies to Mouse Cell-Surface Antigens
4C Monoclonal Antibodies to Rat Leukocyte Surface Antigens, MHC Antigens, and Immunoglobulins

Appendix 5 Suppliers
5 Selected Suppliers of Reagents and Equipment