Current Protocols in Immunology

Last Update: April 11, 2013

Print ISSN: 1934-3671

Online ISSN: 1934-368X

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Chapter 1 Care and Handling of Laboratory Animals
- Introduction
- Section I Animal Health and Husbandry
- Section II Use of Mouse, Rat, Hamster, and Rabbit
- Section III Selected Surgical Procedures
Chapter 2 Induction of Immune Responses
- Introduction
- Section I Assays for Antibody Production
- Section II Production of Antibodies
- Section III Purification and Fragmentation of Antibodies
- Section IV Specialized Techniques
Chapter 3 In Vitro Assays for Mouse Lymphocyte Function
- Introduction
- Section I Isolation and Fractionation of Mononuclear Cell Populations
- Section II Assays for B Cell Function
- Section III Assays for T Cell Function
- Section IV Related Isolation Procedures and Functional Assays
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
- Unit 5.9 Visualization of Multiprotein Complexes by Flow Cytometry
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 γ
- Unit 6.9 Measurement of Antiviral Activity Induced by Interferons α, β, and γ
- Unit 6.10 Measurement of Tumor Necrosis Factor α and β
- Unit 6.11 Measurement of TGF‐β 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
- Unit 6.30 Measurement of Interleukin‐21
Chapter 7 Immunologic Studies in Humans
- Introduction
- Section I Preparation of Human Mononuclear Cell Populations and Subpopulations
- Section II Measurement of Basic Immunologic Characteristics of Human Mononuclear Cells
- Section III Long‐Term and Immortalized Human Cell Lines and Clones
- Section IV Preparation and Functional Analysis of Other Human Lymphoid and Nonlymphoid Cells
Chapter 8 Isolation and Analysis of Proteins
- Introduction
- Section I Solubilization of Cellular Proteins
- Section II Isolation of Proteins Using Antibodies
  - Unit 8.2 Immunoaffinity Chromatography
  - Unit 8.3 Immunoprecipitation
- Section III Electrophoretic Separation of Proteins
- Section IV Detection of 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 9.8 Phage Display Selection, Analysis, and Prediction of B Cell Epitopes
- Unit 9.9 Epitope Mapping Using Gram‐Positive Surface Display
Chapter 10 Molecular Biology
- Introduction
- Section I Isolation and Purification of 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
- Section IV Enzymatic Cleavage and Labeling of DNA
- 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
- Section VII The Polymerase Chain Reaction
Chapter 11 Biochemistry of Cell Activation
- Introduction
- Unit 11.1 Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation
- Unit 11.2 Preparation and Analysis of Phosphorylated Proteins
- Unit 11.3 Detecting Tyrosine‐Phosphorylated Proteins by Western Blot Analysis
- 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 Protein Tyrosine Phosphatase Assays
- 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 Activation and Inhibition of Natural Killer Cells
- 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
- Unit 12.13 Evaluation of B Cell Function in Patients with HIV
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 The Isolation and Characterization of Murine Macrophages
- Unit 14.2 Activation of Murine Macrophages
- Unit 14.3 Characterization of Human Monocytes/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 Fcγ Receptor–Mediated Binding and Phagocytosis
- Unit 14.9 Measurement of Interferon‐Mediated Antiviral Activity of Macrophages
- Unit 14.10 Human Natural Killer Receptors, Co‐Receptors, and Their Ligands
- Unit 14.11 Isolation and Functional Use of Human NKT Cells
- Unit 14.12 Toll‐Like Receptors
- Unit 14.13 Characterization and Functional Analysis of Mouse Invariant Natural T (iNKT) Cells
- Unit 14.14 Measuring Autophagy in Macrophages
- Unit 14.15 Monocyte and Neutrophil Isolation and Migration Assays
- Unit 14.16 Measuring TLR Function in Transfectants
- Unit 14.17 Measurement of Myeloid Cell Immune Suppressive Activity
- Unit 14.18 Measurement of Tumor Cytolysis by Macrophages
- Unit 14.19 In Vivo and Ex Vivo Protocols for Measuring the Killing of Extracellular Pathogens by Macrophages
- Unit 14.20 Measurement of Macrophage Growth and Differentiation
- Unit 14.21 Measurement of Type I Interferon Production
- Unit 14.22 Quantitative Assessment of Macrophage Functions in Repair and Fibrosis
- Unit 14.23 Measuring the Killing of Intracellular Pathogens: Leishmania
- Unit 14.24 Measuring Matrix Metalloproteinase Activity in Macrophages and Polymorphonuclear Leukocytes
- Unit 14.25 Measurement of Macrophage‐Mediated Killing of Intracellular Bacteria, Including Francisella and Mycobacteria
- Unit 14.26 Metabolomics‐Lipidomics of Eicosanoids and Docosanoids Generated by Phagocytes
- Unit 14.27 Measuring Opsonic Phagocytosis via Fcγ Receptors and Complement Receptors on Macrophages
- Unit 14.28 Analysis of Gene Expression and Gene Silencing in Human Macrophages
- Unit 14.29 Target‐Induced Natural Killer Cell Loss as a Measure of NK Cell Responses
- Unit 14.30 Measurement of Phagocytosis, Phagosome Acidification, and Intracellular Killing of Staphylococcus aureus
- Unit 14.31 Measurement of Phagocytic Engulfment of Apoptotic Cells by Macrophages Using pHrodo Succinimidyl Ester
- Unit 14.32 Comparing Individual NK Cell Activity In Vitro
- Unit 14.33 Measuring Intravascular Migration of Mouse Ly6Clow Monocytes In Vivo Using Intravital Microscopy
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, Valvulitis, and Cardiomyopathy
- 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
- Unit 15.21 Creation of “Humanized” Mice to Study Human Immunity
- Unit 15.22 The K/BxN Arthritis Model
- Unit 15.23 Mouse Model for Pyelonephritis
- Unit 15.24 Mouse Models of Atherosclerosis
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
- Unit 16.3 Targeted Identification of Infection‐Related HLA Class I–Presented Epitopes by Stable Isotope Tagging of Epitopes (SITE)
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
- Unit 17.5 Preparation of Stable Single‐Chain Trimers Engineered with Peptide, β2 Microglobulin, and MHC Heavy Chain
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 or Monoclonal Antibody Capture
- 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
- Unit 18.13 Supported Planar Bilayers for Study of the Immunological Synapse
- Unit 18.14 Measurement of Peptide Dissociation from MHC Class II Molecules
- Unit 18.15 Characterizing Protein‐Protein Interactions by Sedimentation Velocity Analytical Ultracentrifugation
- Unit 18.16 Generation of Peptide MHC Class I Monomers and Multimers Through Ligand Exchange
- Unit 18.17 The MHC Motif Viewer: A Visualization Tool for MHC Binding Motifs
- Unit 18.18 Analysis of the Targeting Properties of Fc Chimeric Proteins and Antibodies in Mice In Vivo
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
- Unit 19.13 Cecal Ligation and Puncture
- Unit 19.14 Infection of Mice with Francisella as an Immunological Model
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)
- Unit 20.8 Carcinoembryonic Antigen Transgenic Mouse Models for Immunotherapy and Development of Cancer Vaccines
- Unit 20.9 ErbB2 Transgenic Mice: A Tool for Investigation of the Immune Prevention and Treatment of Mammary Carcinomas
- Unit 20.10 Molecular Cloning and Characterization of MHC Class I– and II–Restricted Tumor Antigens Recognized by T Cells
- Unit 20.11 The Immune Response to Tumors
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
  - Unit 22A.2 Sources of Human and Murine Hematopoietic Stem Cells
- Section B Isolation of Murine Stem and Progenitor Cells
  - Unit 22B.1 Isolation of Murine Hematopoietic Stem Cells and Progenitor Cells
  - Unit 22B.2 Characterization of Mouse Hematopoietic Stem and Progenitor Cells
- Section F Differentiation of Special Populations of Stem and Progenitor Cells
Appendix 1 Abbreviations and Useful Data
- Appendix 1B Amino Acids
- Appendix 1C Commonly Used Mouse Strains
- Appendix 1D Commonly Used Detergents
- Appendix 1E Murine Models of Immunodeficiency and Autoimmune Disease
- Appendix 1F Mouse Endogenous Superantigens: Ms and Mls‐like Determinants Encoded by Mouse Retroviruses
- Appendix 1G HLA Type of EBV‐Transformed Human B Cell Lines
- Appendix 1H Commonly Used Rat Strains
- Appendix 1I Peptide Binding Motifs for MHC Class I and II Molecules
- Appendix 1K Human MHC Genes and Products
- Appendix 1L Mouse MHC Genes and Products
- Appendix 1M Listing, Location, Binding Motifs, and Expression of Nonclassical Class I and Related Genes and Molecules
- Appendix 1N Overview of Protein Folds in the Immune System
- Appendix 1O Nomenclature of the Human T Cell Receptor Genes
- Appendix 1P Nomenclature of the Human Immunoglobulin Genes
- Appendix 1Q Safe Use of Radioisotopes
- Appendix 1R Receptors Involved in the Regulation of Murine NK Cells
- Appendix 1S Nomenclature and Serology of HLA Class I and Class II Alleles
- Appendix 1T Heat‐Shock Proteins
- Appendix 1U Large‐Scale Cell Culture
- Appendix 1V Biosafety: Guidelines for Working with Pathogenic and Infectious Microorganisms
- Appendix 1W Using Bioinformatics Tools for the Sequence Analysis of Immunoglobulins and T Cell Receptors
- Appendix 1X Paired Receptor Systems of the Innate Immune System
Appendix 2 Commonly Used Reagents and Equipment
- Appendix 2A Common Media, Buffers, and Stock Solutions
- Appendix 2B Equipment
Appendix 3 Commonly Used Immunological Techniques
- Appendix 3A Monitoring Cell Growth
- Appendix 3B Trypan Blue Exclusion Test of Cell Viability
- Appendix 3C Wright‐Giemsa and Nonspecific Esterase Staining of Cells
- Appendix 3D Labeling Cells in Microtiter Plates for Determination of [3H]Thymidine Uptake
- Appendix 3E Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures
- Appendix 3F Obtaining Human Peripheral Blood Cells
- Appendix 3G Cryopreservation and Thawing of Cells
- Appendix 3H Dialysis and Concentration of Protein Solutions
- Appendix 3I Column Chromatography of Antibodies and Fragments
- Appendix 3J Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots
- Appendix 3K Silanizing Glassware
- Appendix 3L Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
- Appendix 3M Preparing Frozen Bacterial Stocks
- Appendix 3N Transformation of E. coli by Electroporation
- Appendix 3O Gram Staining
Appendix 4 The CD System of Leukocyte Surface Molecules
- Appendix 4A Monoclonal Antibodies to Human Cell Surface Antigens
- Appendix 4B Monoclonal Antibodies to Mouse Cell‐Surface Antigens
- Appendix 4C Monoclonal Antibodies to Rat Leukocyte Surface Antigens, MHC Antigens, and Immunoglobulins

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