Table of Contents

Last Updated October, 1999

Foreword by Fritz Melchers

1 Care and Handling of Laboratory Animals
2 Antibody Detection and Preparation
3 In Vitro Assays for Mouse Lymphocyte Function
4 In Vivo Assays for Mouse Lymphocyte Function
5 Immunofluorescence and Cell Sorting
6 Cytokines and Their Cellular Receptors
7 Immunologic Studies in Humans
8 Isolation and Analysis of Proteins
9 Peptides
10 Molecular Biology
11 Biochemistry of Cell Activation
12 Detection and Analysis of HIV
13 Complement
14 Macrophages and Monocytes
15 Animal Models for Autoimmune and Inflammatory Disease
16 Antigen Processing and Presentation
17 Engineering Immune Molecules and Receptors
18 Ligand-Receptor Interactions in the Immune System
19 Animal Models of Infectious Diseases
Appendices
Indexes

1 Care and Handling of Laboratory Animals

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

2 Antibody Detection and Preparation

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
2.10 Fragmentation of Immunoglobulin M

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

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 Cells and B Cells
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 In Vitro Antibody Production
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

5 Immunofluorescence and Cell Sorting

6 Cytokines and Their Cellular Receptors

7 Immunologic Studies in Humans

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 T Cell Lines and Clones
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 (New, October 99)

8 Isolation and Analysis of Proteins

8.1 Solubilization of Lymphocytes

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

10 Molecular Biology

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
10.19 Construction of Immunoglobulin Fusion Proteins

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

11 Biochemistry of Cell Activation

12 Detection and Analysis of HIV

13 Complement

14 Macrophages and Monocytes

15 Animal Models for Autoimmune and Inflammatory Disease

16 Antigen Processing and Presentation

17 Engineering Immune Molecules and Receptors

18 Ligand-Receptor Interactions in the Immune System

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
 

Appendices

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. Key Molecules Involved in Receptor-Mediated Lymphocyte Activation
11. Human MHC Genes and Products
12. Mouse MHC Genes and Products
13. Apoptisis Signaling Pathways

A.3 Commonly Used Immunologic Techniques

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. Autoradiography
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. MAb to Human Cell-Surface Antigens
2. MAb to Murine Cell-Surface Antigens
3. MAb to Rat Leukocyte Surface Antigens, MHC Antigens, and Immunoglobulins

 

Indexes