a practical guide to cellular and molecular immunology

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

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 R I - 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

4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA

1194.2.4 Post-hybridization washing & autoradiography

1214.2.5 Autoradiograph development & counter-staining

1234.3 Semi-quantitative RT-PCR to detect cytokine mRNA

1264.3.2 Detection of RT-PCR products

127APPENDICES:

1285.1.3 C3b opsinization of yeast

1325.1.5 Dialysis tubing

1325.1.6 Fixation of tissues for ISH or IHC

1335.1.8 Lysis of red blood cells

1345.1.9 Opsinization of SRBC with antibody

1345.4.10 Protein assay in microtiter plates

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

1.8 Fc R I - 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

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 R I - 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

1204.2.4 Post-hybridization washing & autoradiography

1224.2.5 Autoradiograph development & counter-staining

1244.3 Semi-quantitative RT-PCR to detect cytokine mRNA

1274.3.2 Detection of RT-PCR products

128APPENDICES:

1295.1.3 C3b opsinization of yeast

1335.1.5 Dialysis tubing

1335.1.6 Fixation of tissues for ISH or IHC

1345.1.8 Lysis of red blood cells

1355.1.9 Opsinization of SRBC with antibody

1355.4.10 Protein assay in microtiter plates

150MEM

150RPMI 1640

150RPMI-0% FCS

150RPMI-10% FCS

1505.4 APPENDIX D MAINTENANCE OF CELL LINES

151L-929 cells (for TNF bioassay)

151LM-1 cells (for assay of IL-1)

151Pu5-1.8 cells (macrophage cell line)

1515.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS

1.1 Purification of mononuclear and polymorphonuclear cells 44

1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 47

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 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 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

1234.2.5 Autoradiograph development & counter-staining

1254.3 Semi-quantitative RT-PCR to detect cytokine mRNA

1284.3.2 Detection of RT-PCR products

129APPENDICES:

1305.1.3 C3b opsinization of yeast

1345.1.5 Dialysis tubing

1345.1.6 Fixation of tissues for ISH or IHC

1355.1.8 Lysis of red blood cells

1365.1.9 Opsinization of SRBC with antibody

1365.4.10 Protein assay in microtiter plates

1365.1.11 Splenocytes (single cell suspensions)

151RPMI 1640

151RPMI-0% FCS

151RPMI-10% FCS

1515.4 APPENDIX D MAINTENANCE OF CELL LINES

152L-929 cells (for TNF bioassay)

152LM-1 cells (for assay of IL-1)

152Pu5-1.8 cells (macrophage cell line)

1525.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS

1.1 Purification of mononuclear and polymorphonuclear cells 44

1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 471.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub-populations) 481.2 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 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

123 4.2.5 Autoradiograph development & counter-staining

1254.3 Semi-quantitative RT-PCR to detect cytokine mRNA

1284.3.2 Detection of RT-PCR products

129APPENDICES:

1305.1.3 C3b opsinization of yeast

1345.1.5 Dialysis tubing

1345.1.6 Fixation of tissues for ISH or IHC

1355.1.8 Lysis of red blood cells

136

5.1.8.1 Hypotonic lysis with H2O

1365.1.8.2 Lysis with ammonium chloride

1365.1.9 Opsinization of SRBC with antibody

1365.4.10 Protein assay in microtiter plates

5.1.13.1.2 Giemsa staining of tissue sections

1395.1.13.2 Gills hematoxylin for IHC

1405.1.13.3 Toluidine blue staining (ISH counter-stain)

1405.1.14 Standard curves (e.g., cytokines)

142Actinomycin D

142Alsevers solution

142Ammonium chloride

148Sodium acetate (3M)

14920X SSC (4 liters)

150STE buffer

1505.3 APPENDIX C TISSUE CULTURE MEDIA

152L-929 cells (for TNF bioassay)

152LM-1 cells (for assay of IL-1)

152Pu5-1.8 cells (macrophage cell line)

1525.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS

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

1.8 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 (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

3.6.2 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

4.3 Semi-quantitative RT-PCR to detect cytokine mRNA

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 WORLD WIDE WEB Immunology sites of interest

5.7 APPENDIX G SELECTED TEMPLATES FOR 96 WELL PLATES

1.0 INFLAMMATION: 44

1.1 Purification of mononuclear and polymorphonuclear cells 44

1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 47

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 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 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

1054.1.4 Pre-hybridization, hybridization, and washing

1104.2.1 Probe synthesis & purification

1144.2.2 Preparation of slides for hybridization

1184.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA

1214.2.4 Post-hybridization washing & autoradiography

1234.2.5 Autoradiograph development & counter-staining

1254.3 Semi-quantitative RT-PCR to detect cytokine mRNA

1284.3.2 Detection of RT-PCR products

129APPENDICES:

1305.1.3 C3b opsinization of yeast

134

5.2 APPENDIX B REAGENTS & SOLUTIONS

142Actinomycin D

142Alsevers solution

142Ammonium chloride

142Ammonium sulfate (saturated solutions)

142Borate-buffered saline

142ELISPOT & ELISA Carbonate Coating buffer

142Isotonic Percoll Density Gradient Medium

144PAGE running buffer

144PAGE 2x sample prep buffer

144PAGE gel fix buffer

144Phosphate-buffered saline (PBS)

144Giemsa Stain

144

Giemsa Stock Solution

Reagents for purifying DNA from agarose gels

148General purpose restriction endonuclease buffers

148RNA sample prep buffer (Northern analysis)

148RNA sample dye/loading buffer

148RNAse A

148Salmon sperm DNA

148Sodium acetate (3M)

14920X SSC (4 liters)

150STE buffer

1505.3 APPENDIX C TISSUE CULTURE MEDIA

HBSS (Ca++ and Mg++-free)

151MEM

151RPMI 1640

151RPMI-0% FCS

151RPMI-10% FCS

1515.4 APPENDIX D MAINTENANCE OF CELL LINES

152L-929 cells (for TNF bioassay)

152LM-1 cells (for assay of IL-1)

152Pu5-1.8 cells (macrophage cell line)

1525.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS

1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 481.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub-populations) 491.2 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 purification of IgG antibodies 71

2.2.1 Avid-AL affinity chromatography 712.2.2 Protein A-Sepharose affinity chromatography 732.3 Preparation of IgM antibodies 75

2.4 Analysis & characterization of immunoglobulins 77

2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 772.4.2 Western blotting to detect immunoglobulins 793.0 T CELL AND B CELL RESPONSES: 81

3.1 C'-dependent depletion of CD4+ and CD8+ T Cells 81

3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells 83

3.3 Assessment of T cell proliferation 85

3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells 88

3.5 ELISPOT assays for single cytokine- or Ab-producing cells 90

3.6.1 ELISA assay for detection of antigen-specific antibodies .943.6.2 ELISA assay for detection of cytokines 97

3.8 Immunohistochemical detection of cytokines in tissues

1114.2.1 Probe synthesis & purification

1154.2.2 Preparation of slides for hybridization

119

4.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA

1224.2.4 Post-hybridization washing & autoradiography

1244.2.5 Autoradiograph development & counter-staining

1264.3 Semi-quantitative RT-PCR to detect cytokine mRNA

1294.3.2 Detection of RT-PCR products

130APPENDICES:

1315.1.3 C3b opsinization of yeast

1355.1.5 Dialysis tubing

1355.1.6 Fixation of tissues for ISH or IHC

1365.1.8 Lysis of red blood cells

137

5.1.8.1 Hypotonic lysis with H2O

137

5.1.8.2 Lysis with ammonium chloride

1375.1.9 Opsinization of SRBC with antibody

1405.1.13.2 Gills hematoxylin for IHC

1415.1.13.3 Toluidine blue staining (ISH counter-stain)

1415.1.14 Standard curves (e.g., cytokines)

143Actinomycin D

143

Cesium Chloride (for isolation of total cellular RNA)

RNAse A

149Salmon sperm DNA

149Sodium acetate (3M)

15020X SSC (4 liters)

151STE buffer

1515.3 APPENDIX C TISSUE CULTURE MEDIA

RPMI-10% FCS

1525.4 APPENDIX D MAINTENANCE OF CELL LINES

153L-929 cells (for TNF bioassay)

153LM-1 cells (for assay of IL-1)

153Pu5-1.8 cells (macrophage cell line)

1535.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS

1.1 Purification of mononuclear and polymorphonuclear cells 45

1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 481.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub-populations) 491.2 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 purification of IgG antibodies 71

2.2.1 Avid-AL affinity chromatography 712.2.2 Protein A-Sepharose affinity chromatography 732.3 Preparation of IgM antibodies 75

2.4 Analysis & characterization of immunoglobulins 77

2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 772.4.2 Western blotting to detect immunoglobulins 793.0 T CELL AND B CELL RESPONSES: 81

3.1 C'-dependent depletion of CD4+ and CD8+ T Cells 81

3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells 83

3.3 Assessment of T cell proliferation 85

3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells 88

3.5 ELISPOT assays for single cytokine- or Ab-producing cells 90

3.6.1 ELISA assay for detection of antigen-specific antibodies .943.6.2 ELISA assay for detection of cytokines 97

3.8 Immunohistochemical detection of cytokines in tissues

1114.2.1 Probe synthesis & purification

1154.2.2 Preparation of slides for hybridization

1194.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA

1224.2.4 Post-hybridization washing & autoradiography

1244.2.5 Autoradiograph development & counter-staining

1264.3 Semi-quantitative RT-PCR to detect cytokine mRNA

4.3.2 PCR amplification of the target cDNA

1294.3.2 Detection of RT-PCR products

130APPENDICES:

1315.1.3 C3b opsinization of yeast

1355.1.5 Dialysis tubing

1355.1.6 Fixation of tissues for ISH or IHC

1365.1.8 Lysis of red blood cells

1375.1.9 Opsinization of SRBC with antibody

1375.4.10 Protein assay in microtiter plates

1375.1.11 Splenocytes (single cell suspensions)

1385.1.12 Splenocytes (spleen cell-conditioned medium)

1395.1.13 Staining Protocols

1405.1.14 Standard curves (e.g., cytokines)

1415.1.15 TESPA-treatment of glass slides

153L-929 cells (for TNF bioassay)

153LM-1 cells (for assay of IL-1)

153Pu5-1.8 cells (macrophage cell line)

1535.6 APPENDIX F HUMAN CYTOKINE RT-PCR PRIMERS

1.1 Purification of mononuclear and polymorphonuclear cells 45

1.1a ALTERNATE PROTOCOL 1 (for rapid mononuclear cell gradients) 481.1b ALTERNATE PROTOCOL 2 (fractionation of leukocyte sub-populations) 491.2 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 purification of IgG antibodies 71

2.2.1 Avid-AL affinity chromatography 712.2.2 Protein A-Sepharose affinity chromatography 732.3 Preparation of IgM antibodies 75

2.4 Analysis & characterization of immunoglobulins 77

2.4.1 Polyacrylamide gel electrophoresis of immunoglobulins 772.4.2 Western blotting to detect immunoglobulins 793.0 T CELL AND B CELL RESPONSES: 81

3.1 C'-dependent depletion of CD4+ and CD8+ T Cells 81

3.2 MACS purification (or depletion) of CD4+ and CD8+ T Cells 83

3.3 Assessment of T cell proliferation 85

3.4 Plaque Forming Cell (PFC) assay for IgM-producing cells 88

3.5 ELISPOT assays for single cytokine- or Ab-producing cells 90

3.6.1 ELISA assay for detection of antigen-specific antibodies .943.6.2 ELISA assay for detection of cytokines 97

3.8 Immunohistochemical detection of cytokines in tissues

1114.2.1 Probe synthesis & purification

1154.2.2 Preparation of slides for hybridization

1194.2.3 Hybridization of 35S-cRNA riboprobes to cellular mRNA

1224.2.4 Post-hybridization washing & autoradiography

1244.2.5 Autoradiograph development & counter-staining

1264.3 Semi-quantitative RT-PCR to detect cytokine mRNA

1294.3.2 Detection of RT-PCR products

130APPENDICES:

1315.1.3 C3b opsinization of yeast

1355.1.5 Dialysis tubing

1355.1.6 Fixation of tissues for ISH or IHC

1365.1.8 Lysis of red blood cells

1375.1.9 Opsinization of SRBC with antibody

1375.4.10 Protein assay in microtiter plates

1375.1.11 Splenocytes (single cell suspensions)

1385.1.12 Splenocytes (spleen cell-conditioned medium)

1395.1.13 Staining Protocols

1405.1.14 Standard curves (e.g., cytokines)

1415.1.15 TESPA-treatment of glass slides

1425.2 APPENDIX B REAGENTS & SOLUTIONS