A Practical Guide to Cellular and Molecular Immunology

TABLE OF CONTENTS

1.0 INFLAMMATION
1.1 Purification of mononuclear and polymorphonuclear cells
1.2 Isolation of cells from the peritoneal cavities of mice
1.3 Adherence purification of monocytes
1.4 Antibody- or C3b-dependent phagocytosis
1.5 Activation of macrophages
1.6 Monokine Bioassays
1.6.1 Assay for IL-1 activity
1.6.2 Assay for IL-6 activity
1.6.3 Cytotoxicity assay for TNFá
1.7. Neutrophil chemotaxis assay
1.8. FcåRI-dependent activation of mast cells
2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION
2.1 Hybridoma cell culture with production of monoclonal antibodies
2.2 Affinity purification of IgG antibodies
2.2.1 Avid-AL affinity chromatography
2.2.2 Protein A-Sepharose affinity chromatography
2.3 Preparation of IgM antibodies
2.4 Analysis & characterization of immunoglobulins
2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins
2.4.2 Western blotting to detect immunoglobulins
3.0 T CELL AND B CELL RESPONSES
3.1 C'-dependent depletion of CD4+ and CD8+ T Cells
3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells
3.3 Assessment of T cell proliferation
3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells
3.5 ELISPOT assays for single cytokine- or Ab-producing cells
3.6 ELISA assay for detection of antigen-specific antibodies
3.7 In vivo assessment of T cell responses
3.8 Immunohistochemical detection of cytokines in tissues
4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION
4.1 Northern blotting
4.1.1 Purification of cellular RNA
4.1.2 Electrophoresis of RNA & transfer to membranes
4.1.3 32P-labelled cDNA probe synthesis
4.1.4 Pre-hybridization, hybridization, and washing
4.1.5 Detection of mRNA bands
4.2 In Situ hybridization
4.2.1 Probe synthesis & purification
4.2.2 Preparation of slides for hybridization
4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA
4.2.4 Post-hybridization washing & autoradiography
4.2.5 Autoradiograph development & counter-staining
4.3 Semi-quantitative RT-PCR to detect cytokine mRNA
4.3.1 First strand cDNA Synthesis using Oligo(dT) priming
4.3.2 PCR amplification of the target cDNA
4.3.2 Detection of RT-PCR products


APPENDICES

5.1 APPENDIX A -- GENERAL METHODS
5.2 APPENDIX B -- REAGENTS & SOLUTIONS
5.3 APPENDIX C -- TISSUE CULTURE MEDIA
5.4 APPENDIX D -- MAINTENANCE OF CELL LINES

5.6 APPENDIX F -- Human cytokine RT-PCR primers
5.7 APPENDIX G -- WWW Immunology sites of interest
5.7 APPENDIX G -- Selected templates for 96--well plates

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