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

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



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

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

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

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)


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

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

APPENDICES:

130
5.1 APPENDIX A GENERAL METHODS

130
5.1.1 Anti-sheep RBC antisera

130
5.1.3 C3b opsinization of yeast

134
5.1.5 Dialysis tubing

134
5.1.6 Fixation of tissues for ISH or IHC

135
5.1.8 Lysis of red blood cells

136
5.1.9 Opsinization of SRBC with antibody

136
5.4.10 Protein assay in microtiter plates

136
5.1.11 Splenocytes (single cell suspensions)
12


151
DMEM-0% FCS

151
DMEM-10% FCS.

151
DMEM-10% normal horse serum

151
HBSS (Ca++ and Mg++-free)

151
MEM
13

151
RPMI 1640

151
RPMI-0% FCS.

151
RPMI-10% FCS

151
5.4 APPENDIX D MAINTENANCE OF CELL LINES

152
7TD1 cells (for assay of IL-6)

14
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



129
APPENDICES:

130
5.1 APPENDIX A GENERAL METHODS

130
5.1.1 Anti-sheep RBC antisera

130
5.1.3 C3b opsinization of yeast

134
5.1.5 Dialysis tubing

134
5.1.6 Fixation of tissues for ISH or IHC

135
5.1.8 Lysis of red blood cells

136
5.1.8.1 Hypotonic lysis with H2O

136
5.1.8.2 Lysis with ammonium chloride

136
5.1.9 Opsinization of SRBC with antibody

5.1.15 TESPA-treatment of glass slides

141
5.2 APPENDIX B REAGENTS & SOLUTIONS

142
CELLULAR IMMUNOLOGY REAGENTS

142
Acidified isopropanol

142
Actinomycin D

142
Alsevers solution

142
Ammonium chloride
17

142
Ammonium sulfate (saturated solutions)

142
Borate-buffered saline

142
ELISPOT & ELISA Carbonate Coating buffer



145
Cesium Chloride (for isolation of total cellular RNA)

145
DEPC-treated water (& other solutions)
18

145
Dithiothreitol

145
EDTA (0.5M); pH 8.0

145
Guanidinium Isothiocyanate (GSCN); 5.5 M

145
ISH 10x salts

146
ISH hybridization buffer

147
Northern blotting pre-hyb/hybridization solution

147
MOPS (1 M)

147

150
STE buffer

150
5.3 APPENDIX C TISSUE CULTURE MEDIA

151
Click's medium

151
DMEM

151
DMEM-0% FCS

151
DMEM-10% FCS.

151
DMEM-10% normal horse serum

151
HBSS (Ca++ and Mg++-free)

151
MEM

151
RPMI 1640



155
5.7 APPENDIX G SELECTED TEMPLATES FOR 96 WELL PLATES

157
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
1.8. Fc  RI- 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
21
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


100
4.0 MOLECULAR ANALYSIS OF CYTOKINE mRNA EXPRESSION :

102
4.1 Northern blotting

102
22
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

125

135
5.1.8 Lysis of red blood cells

136
5.1.8.1 Hypotonic lysis with H2O

136
5.1.8.2 Lysis with ammonium chloride

136
5.1.9 Opsinization of SRBC with antibody

136
5.4.10 Protein assay in microtiter plates

136
5.1.11 Splenocytes (single cell suspensions)

137
5.1.12 Splenocytes (spleen cell-conditioned medium)

138
5.1.13 Staining Protocols

139
5.1.13.1 Giemsa stains

139
5.1.13.2 Gills hematoxylin for IHC



142
Borate-buffered saline

142
ELISPOT & ELISA Carbonate Coating buffer

142
Isotonic Percoll Density Gradient Medium

144
PAGE running buffer

144
PAGE 2x sample prep buffer

144
PAGE gel fix buffer

144
Phosphate-buffered saline (PBS)

144
Giemsa Stain

144
25
Giemsa Stock Solution

144

147
Northern blotting pre-hyb/hybridization solution

147
MOPS (1 M)

147
5X MOPS Buffer

147
Phenol (salt-saturated)

147