1 TABLE OF CONTENTS 1.0 INFLAMMATION: 42 1.1 Purification of mononuclear and polymorphonuclear cells 42 1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 45 1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub- populations) 46 1.2 Isolation of cells from the peritoneal cavities of mice 49 1.3 Adherence purification of monocytes 50 1.8. Fc RI- dependent activation of mast cells 65 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 67 2.2 Affinity purification of IgG antibodies 68 2.2.1 Avid-AL affinity chromatography 68 2.2.2 Protein A-Sepharose affinity chromatography 70 2.3 Preparation of IgM antibodies 72 2.4 Analysis & characterization of immunoglobulins 74 2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 74 2.4.2 Western blotting to detect immunoglobulins 76 3.0 T CELL AND B CELL RESPONSES: 78 3.1 C'-dependent depletion of CD4+ and CD8+ T Cells 78 3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells 80 3.3 Assessment of T cell proliferation 82 3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells 85 3.5 ELISPOT assays for single cytokine- or Ab-producing cells 87 3.6.1 ELISA assay for detection of antigen-specific antibodies 91 3.6.2 ELISA assay for detection of cytokines 94 3.8 Immunohistochemical detection of cytokines in tissues 98 4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION : 100 4.1 Northern blotting 100 4.1.2 Electrophoresis of RNA & transfer to membranes 103 4.1.4 Pre-hybridization, hybridization, and washing 108 4.2.1 Probe synthesis & purification 112 4.2.2 Preparation of slides for hybridization 116 2 4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA 119 4.2.4 Post-hybridization washing & autoradiography 121 4.2.5 Autoradiograph development & counter-staining 123 4.3 Semi-quantitative RT-PCR to detect cytokine mRNA 124 4.3.1 First strand cDNA Synthesis using Oligo(dT) priming 125 4.3.2 PCR amplification of the target cDNA 126 4.3.2 Detection of RT-PCR products 127 APPENDICES: 128 5.1 APPENDIX A GENERAL METHODS 128 5.1.1 Anti-sheep RBC antisera 128 5.1.3 C3b opsinization of yeast 132 5.1.5 Dialysis tubing 132 5.1.6 Fixation of tissues for ISH or IHC 133 5.1.8 Lysis of red blood cells 134 5.1.9 Opsinization of SRBC with antibody 134 5.4.10 Protein assay in microtiter plates 3 134 5.1.11 Splenocytes (single cell suspensions) 135 5.1.12 Splenocytes (spleen cell-conditioned medium) 136 5.1.13 Staining Protocols 137 5.1.14 Standard curves (e.g., cytokines) 138 5.1.15 TESPA-treatment of glass slides 139 5.2 APPENDIX B REAGENTS & SOLUTIONS 140 CELLULAR IMMUNOLOGY REAGENTS 140 MOLECULAR BIOLOGY REAGENTS 143 5.3 APPENDIX C TISSUE CULTURE MEDIA 149 Click's medium 149 DMEM 149 DMEM-0% FCS 149 DMEM-10% FCS. 149 DMEM-10% normal horse serum 149 HBSS (Ca++ and Mg++-free) 4 149 MEM 149 RPMI 1640 149 RPMI-0% FCS. 149 RPMI-10% FCS 149 5.4 APPENDIX D MAINTENANCE OF CELL LINES 150 7TD1 cells (for assay of IL-6) 150 Cl.MC/C57.1 cells (C57 mast cells) 150 L-929 cells (for TNF bioassay) 150 LM-1 cells (for assay of IL-1) 150 Pu5-1.8 cells (macrophage cell line) 150 5.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS 152 5.7 APPENDIX G WORLD WIDE WEB Immunology sites of interest 153 5.7 APPENDIX G SELECTED TEMPLATES FOR 96 WELL PLATES 155 1.0 INFLAMMATION: 42 1.1 Purification of mononuclear and polymorphonuclear cells 42 1.2 Isolation of cells from the peritoneal cavities of mice 49 1.3 Adherence purification of monocytes 50 5 1.8. Fc RI- dependent activation of mast cells 65 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 67 2.2 Affinity purification of IgG antibodies 68 2.3 Preparation of IgM antibodies 72 2.4 Analysis & characterization of immunoglobulins 74 3.0 T CELL AND B CELL RESPONSES: 78 3.1 C'-dependent depletion of CD4+ and CD8+ T Cells 78 3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells 80 3.3 Assessment of T cell proliferation 82 3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells 85 3.5 ELISPOT assays for single cytokine- or Ab-producing cells 87 3.6.2 ELISA assay for detection of cytokines 94 3.8 Immunohistochemical detection of cytokines in tissues 98 4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION : 100 4.1 Northern blotting 100 4.3 Semi-quantitative RT-PCR to detect cytokine mRNA 124 APPENDICES: 128 5.1 APPENDIX A GENERAL METHODS 128 5.2 APPENDIX B REAGENTS & SOLUTIONS 140 5.3 APPENDIX C TISSUE CULTURE MEDIA 149 5.4 APPENDIX D MAINTENANCE OF CELL LINES 150 5.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS 152 5.7 APPENDIX G WORLD WIDE WEB Immunology sites of interest 153 5.7 APPENDIX G SELECTED TEMPLATES FOR 96 WELL PLATES 155 6 1.0 INFLAMMATION: 43 1.1 Purification of mononuclear and polymorphonuclear cells 43 1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 46 1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub- populations) 47 1.2 Isolation of cells from the peritoneal cavities of mice 50 1.3 Adherence purification of monocytes 51 1.8. Fc RI- dependent activation of mast cells 66 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 68 2.2 Affinity purification of IgG antibodies 69 2.2.1 Avid-AL affinity chromatography 69 2.2.2 Protein A-Sepharose affinity chromatography 71 2.3 Preparation of IgM antibodies 73 2.4 Analysis & characterization of immunoglobulins 75 2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 75 2.4.2 Western blotting to detect immunoglobulins 77 3.0 T CELL AND B CELL RESPONSES: 79 3.1 C'-dependent depletion of CD4+ and CD8+ T Cells 79 3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells 81 3.3 Assessment of T cell proliferation 83 3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells 86 3.5 ELISPOT assays for single cytokine- or Ab-producing cells 88 3.6.1 ELISA assay for detection of antigen-specific antibodies 92 3.6.2 ELISA assay for detection of cytokines 95 3.8 Immunohistochemical detection of cytokines in tissues 99 4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION : 101 4.1 Northern blotting 101 4.1.2 Electrophoresis of RNA & transfer to membranes 104 4.1.4 Pre-hybridization, hybridization, and washing 109 4.2.1 Probe synthesis & purification 113 4.2.2 Preparation of slides for hybridization 117 4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA 7 120 4.2.4 Post-hybridization washing & autoradiography 122 4.2.5 Autoradiograph development & counter-staining 124 4.3 Semi-quantitative RT-PCR to detect cytokine mRNA 125 4.3.1 First strand cDNA Synthesis using Oligo(dT) priming 126 4.3.2 PCR amplification of the target cDNA 127 4.3.2 Detection of RT-PCR products 128 APPENDICES: 129 5.1 APPENDIX A GENERAL METHODS 129 5.1.1 Anti-sheep RBC antisera 129 5.1.3 C3b opsinization of yeast 133 5.1.5 Dialysis tubing 133 5.1.6 Fixation of tissues for ISH or IHC 134 5.1.8 Lysis of red blood cells 135 5.1.9 Opsinization of SRBC with antibody 135 5.4.10 Protein assay in microtiter plates 8 135 5.1.11 Splenocytes (single cell suspensions) 136 5.1.12 Splenocytes (spleen cell-conditioned medium) 137 5.1.13 Staining Protocols 138 5.1.14 Standard curves (e.g., cytokines) 139 5.1.15 TESPA-treatment of glass slides 140 5.2 APPENDIX B REAGENTS & SOLUTIONS 141 CELLULAR IMMUNOLOGY REAGENTS 141 MOLECULAR BIOLOGY REAGENTS 144 5.3 APPENDIX C TISSUE CULTURE MEDIA 150 Click's medium 150 DMEM 150 DMEM-0% FCS 150 DMEM-10% FCS. 150 DMEM-10% normal horse serum 150 HBSS (Ca++ and Mg++-free) 9 150 MEM 150 RPMI 1640 150 RPMI-0% FCS. 150 RPMI-10% FCS 150 5.4 APPENDIX D MAINTENANCE OF CELL LINES 151 7TD1 cells (for assay of IL-6) 151 Cl.MC/C57.1 cells (C57 mast cells) 151 L-929 cells (for TNF bioassay) 151 LM-1 cells (for assay of IL-1) 151 Pu5-1.8 cells (macrophage cell line) 151 5.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS 153 5.7 APPENDIX G WORLD WIDE WEB Immunology sites of interest 154 5.7 APPENDIX G SELECTED TEMPLATES FOR 96 WELL PLATES 156 1.0 INFLAMMATION: 44 1.1 Purification of mononuclear and polymorphonuclear cells 44 1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 47 10 1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub- populations) 48 1.2 Isolation of cells from the peritoneal cavities of mice 51 1.3 Adherence purification of monocytes 52 1.8. Fc RI- dependent activation of mast cells 67 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 69 2.2 Affinity purification of IgG antibodies 70 2.2.1 Avid-AL affinity chromatography 70 2.2.2 Protein A-Sepharose affinity chromatography 72 2.3 Preparation of IgM antibodies 74 2.4 Analysis & characterization of immunoglobulins 76 2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 76 2.4.2 Western blotting to detect immunoglobulins 78 3.0 T CELL AND B CELL RESPONSES: 80 3.1 C'-dependent depletion of CD4+ and CD8+ T Cells 80 3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells 82 3.3 Assessment of T cell proliferation 84 3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells 87 3.5 ELISPOT assays for single cytokine- or Ab-producing cells 89 3.6.1 ELISA assay for detection of antigen-specific antibodies 93 3.6.2 ELISA assay for detection of cytokines 96 3.8 Immunohistochemical detection of cytokines in tissues 100 4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION : 102 4.1 Northern blotting 102 4.1.2 Electrophoresis of RNA & transfer to membranes 105 4.1.4 Pre-hybridization, hybridization, and washing 110 4.2.1 Probe synthesis & purification 114 4.2.2 Preparation of slides for hybridization 118 4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA 121 4.2.4 Post-hybridization washing & autoradiography [...]... the peritoneal cavities of mice 1.3 Adherence purification of monocytes 1.8 Fc R I - dependent activation of mast cells 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 2.2 Affinity purification of IgG antibodies 2.3 Preparation of IgM antibodies 2.4 Analysis & characterization of immunoglobulins 3.0 T CELL AND B CELL RESPONSES: 3.1 C'-dependent depletion of CD4+ and CD8+ T Cells 3.2 MACS purification... 44 1. 1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 47 1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte subpopulations) 48 1.2 Isolation of cells from the peritoneal cavities of mice 51 14 1.3 Adherence purification of monocytes 52 1.8 Fc R I - dependent activation of mast cells .67 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 69 2.2 Affinity... 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 3.6.2 ELISA assay for detection of cytokines 3.8 Immunohistochemical detection of cytokines in tissues 4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION : 4.1 Northern blotting 21 4.3 Semi-quantitative RT-PCR to detect cytokine... polymorphonuclear cells 44 1. 1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 47 1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte subpopulations) 48 1.2 Isolation of cells from the peritoneal cavities of mice 51 1.3 Adherence purification of monocytes 52 1.8 Fc R I - dependent activation of mast cells .67 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION:... 69 2.2 Affinity purification of IgG antibodies 70 2.2.1 Avid-AL affinity chromatography 70 2.2.2 Protein A- Sepharose affinity chromatography 72 2.3 Preparation of IgM antibodies 74 2.4 Analysis & characterization of immunoglobulins 76 2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 76 2.4.2 Western blotting to detect immunoglobulins 78 3.0 T CELL AND B CELL... 28 1. 1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 48 1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte subpopulations) 49 1.2 Isolation of cells from the peritoneal cavities of mice 52 1.3 Adherence purification of monocytes 53 1.8 Fc R I - dependent activation of mast cells .68 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 70 2.2 Affinity... 45 1. 1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 48 1.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte subpopulations) 49 1.2 Isolation of cells from the peritoneal cavities of mice 52 1.3 Adherence purification of monocytes 53 1.8 Fc R I - dependent activation of mast cells .68 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 70 2.2 Affinity... hybridization 118 4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA 121 4.2.4 Post-hybridization washing & autoradiography 123 4.2.5 Autoradiograph development & counter-staining 125 4.3 Semi-quantitative RT-PCR to detect cytokine mRNA 126 4.3.1 First strand cDNA Synthesis using Oligo(dT) priming 127 4.3.2 PCR amplification... 2.2 Affinity purification of IgG antibodies 70 2.2.1 Avid-AL affinity chromatography 70 2.2.2 Protein A- Sepharose affinity chromatography 72 2.3 Preparation of IgM antibodies 74 2.4 Analysis & characterization of immunoglobulins 76 2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 76 2.4.2 Western blotting to detect immunoglobulins 78 3.0 T CELL AND B CELL RESPONSES:... 2.2 Affinity purification of IgG antibodies 71 2.2.1 Avid-AL affinity chromatography 71 2.2.2 Protein A- Sepharose affinity chromatography 73 2.3 Preparation of IgM antibodies 75 2.4 Analysis & characterization of immunoglobulins 77 2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 77 2.4.2 Western blotting to detect immunoglobulins 79 3.0 T CELL AND B CELL RESPONSES: . antibodies 70 2.2.1 Avid-AL affinity chromatography 70 2.2.2 Protein A- Sepharose affinity chromatography 72 2.3 Preparation of IgM antibodies 74 2.4 Analysis & characterization of immunoglobulins. Post-hybridization washing & autoradiography 11 123 4.2.5 Autoradiograph development & counter-staining 125 4.3 Semi-quantitative RT-PCR to detect cytokine mRNA 126 4.3.1 First strand cDNA. dependent activation of mast cells 2.0 ANTIBODIES: PURIFICATION & CHARACTERIZATION: 2.2 Affinity purification of IgG antibodies 2.3 Preparation of IgM antibodies 2.4 Analysis & characterization