Current Protocols in Immunology
Table of Contents

Foreword by Fritz Melchers
Preface
Contributors

1.1 Animal Health Assurance
1.2 Managing Immunocompromised Animals
1.3 Handling and Restraint
1.4 Anesthesia
1.5 Animal Identification

II USE OF MOUSE, RAT, HAMSTER, AND RABBIT

1.6 Parenteral Injections
1.7 Blood Collection
1.8 Euthanasia

III SELECTED SURGICAL PROCEDURES

1.9 Removal of Lymphoid Organs
1.10 Survival Surgery: Removal of the Spleen or Thymus
1.11 Surgical Removal of Mouse Salivary Glands

2 Antibody Detection and Preparation I ASSAYS FOR ANTIBODY PRODUCTION

2.1 Enzyme-Linked Immunosorbent Assays
2.2 Isotype Determination of Antibodies
2.3 Double-Immunodiffusion Assay for Detecting Specific Antibodies

II PRODUCTION OF ANTIBODIES

2.4 Production of Polyclonal Antisera
2.5 Production of Monoclonal Antibodies
2.6 Monoclonal Antibody Supernatant and Ascites Fluid Production

III PURIFICATION AND FRAGMENTATION OF ANTIBODIES

2.7 Purification of Immunoglobulin G
2.8 Fragmentation of Immunoglobulin G
2.9 Purification of Immunoglobulin M and Immunoglobulin D
2.10A Fragmentation of Immunoglobulin M
2.10B Purification of Human IgA

IV SPECIALIZED TECHNIQUES

2.11 Preparation of Immune Stimulating Complexes (ISCOMs) as Adjuvants
2.12 Cloning, Expression, and Modification of Antibody V Regions 
2.13 Production of Bispecific Antibodies
2.14 Nucleic Acid Immunizations

3 In Vitro Assays for Mouse Lymphocyte Function I ISOLATION AND FRACTIONATION OF MONONUCLEAR CELL POPULATIONS

3.1 Isolation of Mouse Mononuclear Cells
3.2 T Cell Enrichment by Nonadherence to Nylon
3.3 T Cell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells
3.4 T Cell Depletion by Cytotoxic Elimination
3.5A Fractionation of T and B Cells Using Magnetic Beads (Revised, June 2003)
3.5B Isolation of Ly-1⁺/CD5⁺ B Cells by Cell Sorting
3.6 Depletion of Accessory Cells by Adherence to Sephadex G-10
3.7 Isolation of Dendritic Cells

II ASSAYS FOR B CELL FUNCTION

3.8 Assays for B Lymphocyte Function Revised, August 2003
3.9 Early Parameters of B Cell Activation
3.10 Proliferative Assays for B Cell Function

III ASSAYS FOR T CELL FUNCTION

3.11 Induction and Measurement of Cytotoxic T Lymphocyte Activity
3.12 Proliferative Assays for T Cell Function
3.13 Production of T Cell Clones
3.14 Production of Mouse T Cell Hybridomas
3.15 Quantitation of Functional T Cells by Limiting Dilution
3.16 Granule Enzyme Exocytosis Assay for Cytotoxic T Lymphocyte Activation

IV RELATED ISOLATION PROCEDURES AND FUNCTIONAL ASSAYS

3.17 Morphological, Biochemical, and Flow Cytometric Assays of Apoptosis
3.18 Fetal Thymus Organ Culture for T Cell Development Studies
3.19 Isolation of Mouse Small Intestinal Intraepithelial Lymphocytes, Peyer's Patch, and Lamina Propria Cells
3.20 Isolation of Mouse Neutrophils
3.21 Isolation of Mouse Intrahepatic Lymphocytes
3.22 Isolation of Murine Natural Killer Cells

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

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

6 Cytokines and Their Cellular Receptors
6.1 Measurement of Lymphokine Receptors
6.2 Measurement of Soluble and Membrane-Bound Interleukin-1 Using a Fibroblast Bioassay
6.3 Measurement of Human and Murine Interleukin 2 and Interleukin 4
6.4 Measurement of Interleukin 3 and Other Hematopoietic Cytokines
6.5 Measurement of Mouse and Human Interleukin 5
6.6 Measurement of Mouse and Human Interleukin 6
6.7 Measurement of Interleukin 7
6.8 Measurement of Mouse and Human Interferon g
6.9 Measurement of Antiviral Activity Induced by Interferons a, b, and g
6.10 Measurement of Tumor Necrosis Factor a and b
6.11 Measurement of TGF-b in Biological Fluids
6.12 Biological Responses to Chemokine Superfamily Members
6.13 Measurement of Mouse and Human Interleukin 9 
6.14 Measurement of Mouse and Human Interleukin 10
6.15 Measurement of Human Interleukin 11
6.16 Measurement of Human and Mouse Interleukin 12
6.17 Measurement of Human and Murine Stem Cell Factor (c-kit Ligand)
6.18 Measurement of Interleukin 13 
6.19 ELISPOT Assay to Detect Cytokine-Secreting Murine and Human Cells
6.20 Immunoenzymetric Assay of Mouse and Human Cytokines Using NIP-Labeled Anti-Cytokine Antibodies
6.21 Flow Cytometric Detection of Cytokine Receptors
6.22 Measurement of Interleukin 15
6.23 Measurement of Interleukin 16
6.24 Detection of Intracellular Cytokines by Flow Cytometry 
6.25 Measurement of Interleukin 17
6.26 Measurement of Human and Mouse Interleukin 18
6.27 Detection and Isolation of Cytokine Secreting Cells Using the Cytometric Cytokine Secretion Assay
6.28 The In Vivo Cytokine Capture Assay for Measurement of Cytokine Production in the Mouse
6.29 Cytokine, Growth Factor, and Chemokine Ligand Database  

7 Immunologic Studies in Humans
I PREPARATION OF HUMAN MONONUCLEAR CELL POPULATIONS AND SUBPOPULATIONS

7.1 Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood
7.2 Separation of T Cells Using Rosetting Procedures
7.3 Isolation of T Cell Subpopulations
7.4 Immunomagnetic Purification of Specific T Cell Subpopulations
7.5 Isolation of B Cell Populations
7.6 Isolation of Monocyte/Macrophage Populations
7.7 Isolation of Cell Populations Containing NK Cells and LAK Precursor Cells
7.8 Isolation of Mononuclear Cells from Tonsillar Tissue
7.9 Detection of Unseparated Human Lymphocytes by Flow Cytometry

II MEASUREMENT OF BASIC IMMUNOLOGIC CHARACTERISTICS OF HUMAN MONONUCLEAR CELLS

7.10 Measurement of Proliferative Responses of Cultured Lymphocytes
7.11 Induction of Polyclonal Immunoglobulin Responses
7.12 Measurement of Polyclonal Immunoglobulin Synthesis Using ELISA
7.13 Measurement of Polyclonal Immunoglobulin Synthesis Using Reverse Plaque Technique
7.14 Measurement of Polyclonal Immunoglobulin Synthesis Using the ELISPOT Assay
7.15 Measurement of In Vitro Specific-Antibody Synthesis
7.16 Induction and Measurement of In Vivo Antibody Responses
7.17 Measurement of Polyclonal and Antigen-Specific Cytotoxic T Cell Function
7.18 Measurement of Cytotoxic NK/LAK Cells

III LONG-TERM AND IMMORTALIZED HUMAN CELL LINES AND CLONES

7.19 Generation and Maintenance of Cloned Human T Cell Lines
7.20 Generation of HTLV-I-Transformed T Cell Lines
7.21 Generation of T Cell Hybridomas
7.22 Generation of Epstein-Barr (EBV)-Immortalized B Cell Lines

IV PREPARATION AND FUNCTIONAL ANALYSIS OF HUMAN NONLYMPHOID CELLS

7.23 Isolation and Functional Analysis of Neutrophils
7.24 Isolation of Human Basophils
7.25 Isolation of Tissue Mast Cells
7.26 Measuring Degranulation of Mast Cells
7.27 ⁵¹Cr Release Assay of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)
7.28 Measurement of Cellular Adhesion Under Static Conditions
7.29 Cell Adhesion Under Hydrodynamic Flow Conditions
7.30 Isolation of Human Intestinal Mucosal Mononuclear Cells
7.31 Isolation of Human Eosinophils
7.32 Isolation and Generation of Human Dendritic Cells
7.33 Measurement of Prostaglandins and Other Eicosanoids
7.34 Isolation and Characterization of Human Natural Killer Cell Subsets HTML PDF (Size 228KB)
7.35 Assays for B Cell and Germinal Center Development

8 Isolation and Analysis of Proteins

I SOLUBILIZATION OF CELLULAR PROTEINS

8.1A Solubilization of Lymphocytes
8.1B Purification of Organelles from Mammalian Cells(August 2003)

II ISOLATION OF PROTEINS USING ANTIBODIES

8.2 Immunoaffinity Chromatography
8.3 Immunoprecipitation

III ELECTROPHORETIC SEPARATION OF PROTEINS

8.4 One-Dimensional Gel Electrophoresis of Proteins
8.5 Two-Dimensional Gel Electrophoresis Using the O'Farrell System
8.6 Diagonal Gel Electrophoresis
8.7 Isolation of Proteins for Microsequence Analysis
8.8 Electroelution of Proteins from Stained Gels

IV DETECTION OF PROTEINS

8.9 Staining Proteins in Gels
8.10 Immunoblotting and Immunodetection
8.11 Iodination of Soluble and Membrane-Bound Proteins and Cells
8.12 Biosynthetic Labeling of Proteins
8.13 Metabolic Radiolabeling of Animal Cell Glycoconjugates
8.14 Inhibition of N-Linked Glycosylation
8.15 Release of Saccharides from Glycoconjugates
8.16 Biotinylation and Analysis of Membrane-Bound and Soluble Proteins

9 Peptides
9.1 Introduction to Peptide Synthesis 
9.2 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins (Revised, June 2003)
9.3 Production of Anti-Peptide Antisera (Revised, June 2003) 9.4 Production of Antipeptide Antisera
9.5 Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries 
9.6 Synthesis and Use of Multiple Antigen Peptide (MAP) Systems
9.7 Synthesis of Peptides Immobilized on Polypropylene Pins
9.8 Phage Display Selection and Analysis of Ab-Binding Epitopes 

10 Molecular Biology
I ISOLATION AND PURIFICATION OF DNA

10.1 Purification and Concentration of DNA from Aqueous Solutions
10.2 Preparation of Genomic DNA from Mammalian Tissue

10.3 Preparation of Bacterial Plasmid DNA

II RESOLUTION AND RECOVERY OF DNA FRAGMENTS

10.4 Agarose Gel Electrophoresis
10.5 Isolation and Purification of Large DNA Restriction Fragments from Agarose Gels

III ANALYSIS OF GENOMIC DNA BY BLOTTING AND HYBRIDIZATION

10.6A Southern Blotting
10.6B Hybridization Analysis of DNA Blots
10.7 Deprotection of Oligonucleotides and Purification Using Denaturing PAGE

IV ENZYMATIC CLEAVAGE AND LABELING OF DNA

10.8 Digestion of DNA with Restriction Endonucleases
10.9 Reagents and Radioisotopes Used to Manipulate Nucleic Acids
10.10 Enzymatic Labeling of Nucleic Acids

V PREPARATION AND ANALYSIS OF RNA

10.11 Preparation of RNA from Tissues and Cells
10.12 Analysis of RNA by Northern and Slot-Blot Hybridization

VI TRANSFECTION AND EXPRESSION OF CLONED DNA

10.13 Calcium Phosphate Transfection
10.14 Transfection Using DEAE-Dextran
10.15 Transfection by Electroporation
10.16 Liposome-Mediated Transfection
10.17A Stable Transfer of Genes into Mammalian Cells
10.17B Transfection of Lymphoid Cells
10.17C Rapid Production of Retroviruses Using 293T Cell-Based Systems
10.18 Use of Monoclonal Antibodies for Expression Cloning
10.19A Construction of Immunoglobulin Fusion Proteins
10.19B Phage Display of Single-Chain Antibody Constructs

VII THE POLYMERASE CHAIN REACTION

10.20 Enzymatic Amplification of DNA by PCR
10.21 Quantitation of Rare DNAs by PCR
10.22 Detection of Isotype Switch Rearrangement in Bulk Culture by PCR
10.23 Detection of Cytokine mRNA Expression by PCR
10.24 cDNA Amplification Using One-Sided (Anchored) PCR
10.25 Double-Stranded DNA Sequence Analysis
10.26 Evaluating T Cell Receptor Gene Expression by PCR
10.27 Molecular Analysis of Mouse T Cell Receptor Expression Using PCR
10.28 Spectratype/Immunoscope Analysis of the Expressed TCR Repertoire
10.29 Multiprobe Ribonuclease Protection Assay for Simultaneous Measurement of mRNA Expression 
10.30 Analysis of Telomere Length and Telomerase Activity
10.31 Measurement of Mouse T Cell Receptor Excision Circles

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

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

13 Complement
13.1 Classical Pathway Evaluation
13.2 Alternative Pathway Evaluation
13.3 Isolation and Purification of C3 from Human Plasma
13.4 Assays for Membrane Complement Receptors
13.5 Isolation and Functional Assay of the Membrane Complement Inhibitors CD55 (DAF) and CD59 (MIRL)
13.6 Assays for the Mannan-Binding Lectin Pathway

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

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

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

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

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

19 Animal Models of Infectious Diseases
19.1 Schistosomiasis
19.2 Animal Models for the Analysis of Immune Responses to Leishmaniasis
19.3 Animal Models for Toxopolasma gondii Infection
19.4 Experimental Asexual Blood Stage Malaria Immunity
19.5 Animal Models of Mycobacteria Infection
19.6 Animal Models of Candidiasis
19.7 A Mouse Model for Cytomegalovirus Infection
19.8 Helicobacter Animal Models
19.9 Animal Model for Infection with Listeria monocytogenes
19.10 Animal Models Using Lymphocytic Choriomeningitis Virus
19.11 Influenza Virus
19.12 Animal Model of Nippostrongylus Brasiliensis and Heligmosomoides Polygyrus (New, June 2003)
 20 Tumor Immunology
20.1 B16 as a Mouse Model for Human Melanoma
20.2 Mouse 4T1 Breast Tumor Model
20.3 Mouse SaI Sarcoma Tumor Model
20.4 The P815 Mastocytoma Tumor Model
20.5 The TRAMP Mouse as a Model for Prostate Cancer 
20.6 Models for Lymphoma 

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

Appendices A.1 Abbreviations and Useful Data

1. Abbreviations Used in This Manual 
2. Amino Acids 
3. Commonly Used Mouse Strains 
4. Commonly Used Detergents 
5. Murine Models of Immunodeficiency and Autoimmune Disease 
6. Mouse Endogenous Superantigens: Mls and Mls-like Determinants Encoded by Mouse Retroviruses 
7. HLA Type of EBV-Transformed Human B Cell Lines 
8. Commonly Used Rat Strains I Peptide Binding Motifs for MHC Class I and II Molecules 
9. Peptide-Binding Motifs for MHC Class I and II Molecules 
10. Web Sites of Interest to Immunologists 
11. Human MHC Genes and Products 
12. Mouse MHC Genes and Products 
13. Listing, Location, Binding, Motifs, and Expression of Nonclassical Class I and Related Genes and Molecules 
14. Overview of Protein Folds in the Immune System 
15. Nomenclature of the Human T Cell Receptor Genes 
16. Nomenclature of the Human Immunoglobulin Genes 
17. Safe Use of Radioisotopes
18. Receptors Involved in the Regulation of Murine NK Cells
19. Nomenclature and Serology of HLA Class I and Class II Alleles
    
20. Nomenclature and Serology of HLA Class I and Class II Alleles
    
21. Large-Scale Cell Culture

1. Common Media, Buffers, and Stock Solutions 
2. Equipment

1. Monitoring Cell Growth 
2. Trypan Blue Exclusion Test of Cell Viability 
3. Wright-Giemsa and Nonspecific Staining 
4. Labeling Cells with Microtiter Plates for Determination of [³H]Thymidine 
5. Diagnosis and Treatment of Mycoplasma-Contaminated Cell Cultures 
6. Obtaining Human Peripheral Blood Cells 
7. Cryopreservation of Cells 
8. Dialysis and Concentration of Protein Solutions 
9. Column Chromatography of Antibodies and Fragments 
10.  Detection and Quantitation of Radiolabeled Proteins and DNA in Gels and Blots 
11. Silanizing Glassware 
12. Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy 
13. Preparing Frozen Bacterial Stocks 
14. Transformation of E. coli by Electroporation 
15. Gram Staining

1. Monoclonal Antibodies to Human Cell Surface Antigens 
2. Monoclonal Antibodies to Mouse Cell-Surface Antigens 
3. MAb to Rat Leukocyte Surface Antigens, MHC Antigens, and Immunoglobulins

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