Cytokine cell biology : a practical approach /
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ISBN：9780199638598
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Summary:
Publisher Summary 1
A laboratory guide to current cytokine research methodology, focusing on cell biology techniques for studying cytokines, cytokine receptors, and cytokine driven processes. Chapters cover areas including human B- cell responses to cytokines, T cells and cytokines, the generation and quantitation of cell-mediated cytotoxicity, cytokine regulation of endothelial cells, assays for leucocyte migration, flow cytometric detection of intracellular cytokines, development of antibodies to cytokines, and quantitative biological assays. This third edition contains new information reflecting progress in the field since 1995. Balkwill is affiliated with St. Bartholomews and the Royal London Hospital School of Medicine and Dentistry, UK. Annotation c. Book News, Inc., Portland, OR (booknews.com)
目录
List of protocols p. xv
Abbreviations p. xxi
Measurement of proliferative, cytostatic, and cytolytic activity of cytokines p. 1
Introduction p. 1
Effects of cytokines on cell growth in vitro p. 1
Direct cell counting p. 2
Determination of the effects on growth of cytokines using indirect methods p. 5
The alkaline phosphatase assay p. 5
The MTT-type assay p. 6
Measurement of cell lysis using crystal violet p. 6
Cell-cycle studies p. 7
Measurement of [superscript 3H]thymidine incorporation into acid-insoluble material p. 7
Autoradiography of labelled nuclei p. 9
Cytofluorimetry p. 10
Measurement of bromodeoxyuridine incorporation to assess cell-cycle changes in vivo p. 11
Mitotic index p. 13
DNA fragmentation p. 14
References p. 16
Human B-cell responses to cytokines p. 17
Introduction p. 17
Reagents used for in vitro experiments with human B cells p. 18
Culture media p. 18
Holding medium p. 19
Lymphocyte separation medium p. 19
Sheep red blood cells (SRBC) p. 20
Phorbol ester p. 20
Anti-IgM p. 20
Staphylococcus aureus Cowan 1 strain (SAC) p. 21
Influenza virus p. 21
[superscript 3H]thymidine p. 21
Antibodies for labelling B cells p. 21
CD40 antibody and CD40L p. 21
Recombinant cytokines p. 22
Preparation of human B cells p. 22
Mononuclear cell preparation p. 23
Depletion of T cells by E-rosetting p. 24
Depletion of monocytes by adherence p. 25
Preparation of heavy and light B cells p. 27
Separation of B-cell subpopulations with monoclonal antibodies p. 27
Immunofluorescence analysis of B-cell preparations p. 29
Human B cell-lines for assaying B-cell growth and differentiation factors p. 32
B-cell activation p. 33
Measurement of RNA synthesis p. 33
Measurement of B-cell activation by expression of surface activation antigens such as CD23 and surface IgM p. 34
Assays for B-cell proliferation in response to cytokines p. 36
Cytokine stimulation of B-cell proliferation p. 36
Cytokine stimulation of proliferation by B cell-lines p. 38
Assays for cytokines that stimulate human B-cell differentiation (Ig secretion) p. 39
Co-stimulation assays for cytokines that stimulate immunoglobulin secretion (B-cell differentiation factors) p. 39
Assays for B-cell differentiation factors using B cell-lines p. 40
Assays for cytokine regulation of Ig isotype responses p. 40
T-cell replacing factors p. 42
Enzyme immunoassays for immunoglobulin secreted by human B cells p. 43
Measurement of Ig (IgM, IgG, IgA, and IgE) secretion p. 43
Measurement of IgG subclasses by ELISA p. 46
ELISA for specific antibody production p. 46
References p. 48
T cells and cytokines p. 51
Introduction p. 51
Isolation of T cells for the analysis of cytokine production p. 52
Isolation of lymphocytes using a one-step Lymphoprep gradient p. 52
Positive selection of T cells p. 56
Isolation of naive CD4 T cells (CD4+CD45RA+) and CD8 T cells (CD8+CD45RA+) p. 57
Analysis of T-cell cytokine production p. 57
Cytokine production by T cells of different phenotypes p. 60
Cytokine production by CD4 T cells p. 60
Cytokine production by CD8 T cells p. 61
Cytokine production by double-negative (CD4-CD8-) [alpha beta] T cells p. 62
Cytokine production by [gamma delta] T cells p. 62
Cytokine production by antigen-specific T cells p. 63
Effect of cytokines on T cells p. 66
Conclusions p. 67
References p. 67
The generation and quantitation of cell-mediated cytotoxicity p. 69
Introduction p. 69
Techniques for the isolation of lymphocyte populations p. 71
Isolation of PBMC, PBL, or LGL p. 71
Isolation of specific lymphoid populations based upon phenotypic markers or light-scatter characteristics p. 73
Isolation of tumour-infiltrating lymphocytes p. 75
Generation of lymphokine-activated killers p. 76
The generation of IL-2 activated killers p. 76
Anti-CD3/IL-2 activated 'T-cell' killers p. 77
Alternative cytokine activation strategies p. 78
Cryopreservation of lymphocytes and tumours p. 78
Cytotoxicity assays p. 79
[superscript 51]Cr-release cytotoxicity assay p. 80
Single-cell CMC assay p. 82
Alternative approaches for measuring CMC, including non-radioactive assays p. 83
References p. 84
Cytokine regulation of endothelial cells p. 87
Introduction p. 87
Endothelial cells p. 88
Human umbilical venous EC p. 88
Mouse PmT-transformed lines p. 89
Chemotaxis p. 91
Boyden chamber assay p. 92
Microchemotaxis assay p. 93
Leucocyte adhesion and transmigration p. 94
Adhesion p. 95
Transmigration p. 96
Reverse transmigration p. 98
The air-pouch model p. 99
Comments p. 100
References p. 100
Assays for leucocyte migration p. 103
Introduction p. 103
Preparation of target cells and test samples p. 104
Isolation of leucoyte subsets for chemotaxis p. 104
Preparation of test samples and standards for chemotaxis p. 106
Chemotaxis under agarose p. 106
Chemotaxis through micropore filters p. 110
In vivo leucoyte migration in response to cytokines p. 112
Acknowledgements p. 114
References p. 114
Biological assays for haemopoietic growth factors p. 115
Introduction p. 115
Preparation of cell suspensions p. 116
Murine bone marrow p. 116
Human bone marrow p. 117
Separation of excess red cells from human bone-marrow samples p. 118
Standard clonal assays p. 118
Murine bone marrow p. 118
Human bone marrow or peripheral blood p. 120
General comments p. 120
Serum-free cultures p. 121
General comments p. 121
Preparation of the reagents p. 121
Preparation of serum-free medium p. 121
Comments p. 122
Growth factor-dependent cell lines p. 122
Enrichment of progenitor-cell populations from murine bone marrow p. 124
General comments p. 124
Murine cells p. 126
Human cells p. 129
References p. 134
Assays for macrophage activation by cytokines p. 137
Introduction p. 137
Assay for respiratory-burst activity p. 138
Comments p. 139
Assay for the secretion of nitric oxide p. 139
Comments p. 140
MHC class II expression p. 140
Comments p. 141
Macrophage mannosyl receptor (MMR) assays p. 142
Single-cell assays p. 144
Macrophage cytokine assays p. 145
Conclusions p. 145
References p. 146
Cytokine production by individual cells p. 147
Introduction p. 147
Immunolocalization of cytokines p. 148
Immunostaining techniques for the localization of cytokines in cytocentrifuge preparations of human or murine cells p. 148
Non-isotopic in situ hybridization for the detection of cytokine mRNA in wax-embedded tissue sections p. 155
Detection of cytokine release by individual cells p. 157
Reverse haemolytic plaque assay (RHPA) p. 157
Cell-blot assay p. 162
Elispot assay p. 165
Discussion p. 166
Acknowledgements p. 168
References p. 168
Flow cytometric detection of intracellular cytokines p. 171
Introduction p. 171
Technique background p. 171
Experimental advantages and disadvantages p. 172
Cell activation p. 172
Polyclonal activators p. 172
Physiological activators p. 174
Considerations when surface staining p. 174
Fixation p. 175
Intracellular staining p. 177
Permeabilization p. 177
Cytokine staining p. 177
References p. 180
Development of antibodies to cytokines p. 181
Introduction p. 181
Choice of immunogen p. 181
Recombinant cytokines p. 181
Purified 'natural' cytokines p. 181
Conjugates of cytokines p. 182
Small peptide homologues p. 182
Enzymatic digests of cytokines p. 186
Choice of animal p. 187
The adjuvant p. 187
Immunization schedules p. 188
Mice p. 188
Rabbits p. 188
Guinea-pigs p. 189
Sheep and goats p. 190
Screening p. 190
Purification of antibodies p. 191
Acknowledgements p. 192
References p. 192
RIA, IRMA, and ELISA for cytokines and their soluble receptors p. 193
Introduction p. 193
Radioimmunoassays (RIA) p. 194
Immunoradiometric assays (IRMA) p. 196
Enzyme-limked immunosorbent assays (ELISA) p. 200
Problems p. 203
Calibration p. 203
Mixtures of cytokine and soluble receptors p. 203
Assay diluent or matrix p. 205
Samples from different sources p. 205
References p. 206
Quantitative biological assays p. 207
Introduction p. 207
New bioassays p. 210
Bioassays compared with immunoassays and receptor binding assays p. 210
Biological assays for interleukins p. 210
Bioassay for interleukin-2 (IL-2) p. 210
Bioassay for interleukin-1 (IL-1) p. 212
Bioassay for interleukin-4 (IL-4) p. 213
Bioassay for interleukin-5 (IL-5) p. 214
Bioassay for interleukin-6 (IL-6) p. 214
Bioassay for interleukin-7 (IL-7) p. 215
Bioassay for interleukin-8 (IL-8) and other chemokines p. 216
Bioassay for interleukin-9 (IL-9) p. 216
Bioassay for interleukin-10 (IL-10) p. 217
Bioassay for interleukin-11 (IL-11) p. 218
Bioassay for interleukin-12 (IL-12) p. 219
Bioassay for interleukin-13 (IL-13) p. 219
Bioassay for interleukin-14 (IL-14) p. 220
Bioassay for interleukin-15 (IL-15) p. 221
Bioassay for interleukin-16 (IL-16) p. 221
Bioassay for interleukin-17 (IL-17) p. 221
Bioassay for interleukin-18 (IL-18) p. 222
Bioassays for colony-stimulating factors--interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem-cell factor (SCF), thrombopoietin (TPO), and leukaemia inhibitory factor (LIF) p. 223
Introduction p. 223
Bioassay for leukaemia inhibitory factor (LIF) p. 227
Bioassay for thrombopoietin (TPO) p. 227
Bioassays for TGF-[beta] and related proteins p. 228
Bioassay for TGF-[beta] p. 228
Bone morphogenetic proteins p. 229
Bioassays for other cytokines p. 231
Bioassays for tumour necrosis factors p. 231
Bioassay for interferons (IFNs) p. 232
Bioassay for oncostatin-M p. 234
Analysis of results p. 235
Acknowledgements p. 238
References p. 238
List of suppliers p. 241
Index p. 251
Abbreviations p. xxi
Measurement of proliferative, cytostatic, and cytolytic activity of cytokines p. 1
Introduction p. 1
Effects of cytokines on cell growth in vitro p. 1
Direct cell counting p. 2
Determination of the effects on growth of cytokines using indirect methods p. 5
The alkaline phosphatase assay p. 5
The MTT-type assay p. 6
Measurement of cell lysis using crystal violet p. 6
Cell-cycle studies p. 7
Measurement of [superscript 3H]thymidine incorporation into acid-insoluble material p. 7
Autoradiography of labelled nuclei p. 9
Cytofluorimetry p. 10
Measurement of bromodeoxyuridine incorporation to assess cell-cycle changes in vivo p. 11
Mitotic index p. 13
DNA fragmentation p. 14
References p. 16
Human B-cell responses to cytokines p. 17
Introduction p. 17
Reagents used for in vitro experiments with human B cells p. 18
Culture media p. 18
Holding medium p. 19
Lymphocyte separation medium p. 19
Sheep red blood cells (SRBC) p. 20
Phorbol ester p. 20
Anti-IgM p. 20
Staphylococcus aureus Cowan 1 strain (SAC) p. 21
Influenza virus p. 21
[superscript 3H]thymidine p. 21
Antibodies for labelling B cells p. 21
CD40 antibody and CD40L p. 21
Recombinant cytokines p. 22
Preparation of human B cells p. 22
Mononuclear cell preparation p. 23
Depletion of T cells by E-rosetting p. 24
Depletion of monocytes by adherence p. 25
Preparation of heavy and light B cells p. 27
Separation of B-cell subpopulations with monoclonal antibodies p. 27
Immunofluorescence analysis of B-cell preparations p. 29
Human B cell-lines for assaying B-cell growth and differentiation factors p. 32
B-cell activation p. 33
Measurement of RNA synthesis p. 33
Measurement of B-cell activation by expression of surface activation antigens such as CD23 and surface IgM p. 34
Assays for B-cell proliferation in response to cytokines p. 36
Cytokine stimulation of B-cell proliferation p. 36
Cytokine stimulation of proliferation by B cell-lines p. 38
Assays for cytokines that stimulate human B-cell differentiation (Ig secretion) p. 39
Co-stimulation assays for cytokines that stimulate immunoglobulin secretion (B-cell differentiation factors) p. 39
Assays for B-cell differentiation factors using B cell-lines p. 40
Assays for cytokine regulation of Ig isotype responses p. 40
T-cell replacing factors p. 42
Enzyme immunoassays for immunoglobulin secreted by human B cells p. 43
Measurement of Ig (IgM, IgG, IgA, and IgE) secretion p. 43
Measurement of IgG subclasses by ELISA p. 46
ELISA for specific antibody production p. 46
References p. 48
T cells and cytokines p. 51
Introduction p. 51
Isolation of T cells for the analysis of cytokine production p. 52
Isolation of lymphocytes using a one-step Lymphoprep gradient p. 52
Positive selection of T cells p. 56
Isolation of naive CD4 T cells (CD4+CD45RA+) and CD8 T cells (CD8+CD45RA+) p. 57
Analysis of T-cell cytokine production p. 57
Cytokine production by T cells of different phenotypes p. 60
Cytokine production by CD4 T cells p. 60
Cytokine production by CD8 T cells p. 61
Cytokine production by double-negative (CD4-CD8-) [alpha beta] T cells p. 62
Cytokine production by [gamma delta] T cells p. 62
Cytokine production by antigen-specific T cells p. 63
Effect of cytokines on T cells p. 66
Conclusions p. 67
References p. 67
The generation and quantitation of cell-mediated cytotoxicity p. 69
Introduction p. 69
Techniques for the isolation of lymphocyte populations p. 71
Isolation of PBMC, PBL, or LGL p. 71
Isolation of specific lymphoid populations based upon phenotypic markers or light-scatter characteristics p. 73
Isolation of tumour-infiltrating lymphocytes p. 75
Generation of lymphokine-activated killers p. 76
The generation of IL-2 activated killers p. 76
Anti-CD3/IL-2 activated 'T-cell' killers p. 77
Alternative cytokine activation strategies p. 78
Cryopreservation of lymphocytes and tumours p. 78
Cytotoxicity assays p. 79
[superscript 51]Cr-release cytotoxicity assay p. 80
Single-cell CMC assay p. 82
Alternative approaches for measuring CMC, including non-radioactive assays p. 83
References p. 84
Cytokine regulation of endothelial cells p. 87
Introduction p. 87
Endothelial cells p. 88
Human umbilical venous EC p. 88
Mouse PmT-transformed lines p. 89
Chemotaxis p. 91
Boyden chamber assay p. 92
Microchemotaxis assay p. 93
Leucocyte adhesion and transmigration p. 94
Adhesion p. 95
Transmigration p. 96
Reverse transmigration p. 98
The air-pouch model p. 99
Comments p. 100
References p. 100
Assays for leucocyte migration p. 103
Introduction p. 103
Preparation of target cells and test samples p. 104
Isolation of leucoyte subsets for chemotaxis p. 104
Preparation of test samples and standards for chemotaxis p. 106
Chemotaxis under agarose p. 106
Chemotaxis through micropore filters p. 110
In vivo leucoyte migration in response to cytokines p. 112
Acknowledgements p. 114
References p. 114
Biological assays for haemopoietic growth factors p. 115
Introduction p. 115
Preparation of cell suspensions p. 116
Murine bone marrow p. 116
Human bone marrow p. 117
Separation of excess red cells from human bone-marrow samples p. 118
Standard clonal assays p. 118
Murine bone marrow p. 118
Human bone marrow or peripheral blood p. 120
General comments p. 120
Serum-free cultures p. 121
General comments p. 121
Preparation of the reagents p. 121
Preparation of serum-free medium p. 121
Comments p. 122
Growth factor-dependent cell lines p. 122
Enrichment of progenitor-cell populations from murine bone marrow p. 124
General comments p. 124
Murine cells p. 126
Human cells p. 129
References p. 134
Assays for macrophage activation by cytokines p. 137
Introduction p. 137
Assay for respiratory-burst activity p. 138
Comments p. 139
Assay for the secretion of nitric oxide p. 139
Comments p. 140
MHC class II expression p. 140
Comments p. 141
Macrophage mannosyl receptor (MMR) assays p. 142
Single-cell assays p. 144
Macrophage cytokine assays p. 145
Conclusions p. 145
References p. 146
Cytokine production by individual cells p. 147
Introduction p. 147
Immunolocalization of cytokines p. 148
Immunostaining techniques for the localization of cytokines in cytocentrifuge preparations of human or murine cells p. 148
Non-isotopic in situ hybridization for the detection of cytokine mRNA in wax-embedded tissue sections p. 155
Detection of cytokine release by individual cells p. 157
Reverse haemolytic plaque assay (RHPA) p. 157
Cell-blot assay p. 162
Elispot assay p. 165
Discussion p. 166
Acknowledgements p. 168
References p. 168
Flow cytometric detection of intracellular cytokines p. 171
Introduction p. 171
Technique background p. 171
Experimental advantages and disadvantages p. 172
Cell activation p. 172
Polyclonal activators p. 172
Physiological activators p. 174
Considerations when surface staining p. 174
Fixation p. 175
Intracellular staining p. 177
Permeabilization p. 177
Cytokine staining p. 177
References p. 180
Development of antibodies to cytokines p. 181
Introduction p. 181
Choice of immunogen p. 181
Recombinant cytokines p. 181
Purified 'natural' cytokines p. 181
Conjugates of cytokines p. 182
Small peptide homologues p. 182
Enzymatic digests of cytokines p. 186
Choice of animal p. 187
The adjuvant p. 187
Immunization schedules p. 188
Mice p. 188
Rabbits p. 188
Guinea-pigs p. 189
Sheep and goats p. 190
Screening p. 190
Purification of antibodies p. 191
Acknowledgements p. 192
References p. 192
RIA, IRMA, and ELISA for cytokines and their soluble receptors p. 193
Introduction p. 193
Radioimmunoassays (RIA) p. 194
Immunoradiometric assays (IRMA) p. 196
Enzyme-limked immunosorbent assays (ELISA) p. 200
Problems p. 203
Calibration p. 203
Mixtures of cytokine and soluble receptors p. 203
Assay diluent or matrix p. 205
Samples from different sources p. 205
References p. 206
Quantitative biological assays p. 207
Introduction p. 207
New bioassays p. 210
Bioassays compared with immunoassays and receptor binding assays p. 210
Biological assays for interleukins p. 210
Bioassay for interleukin-2 (IL-2) p. 210
Bioassay for interleukin-1 (IL-1) p. 212
Bioassay for interleukin-4 (IL-4) p. 213
Bioassay for interleukin-5 (IL-5) p. 214
Bioassay for interleukin-6 (IL-6) p. 214
Bioassay for interleukin-7 (IL-7) p. 215
Bioassay for interleukin-8 (IL-8) and other chemokines p. 216
Bioassay for interleukin-9 (IL-9) p. 216
Bioassay for interleukin-10 (IL-10) p. 217
Bioassay for interleukin-11 (IL-11) p. 218
Bioassay for interleukin-12 (IL-12) p. 219
Bioassay for interleukin-13 (IL-13) p. 219
Bioassay for interleukin-14 (IL-14) p. 220
Bioassay for interleukin-15 (IL-15) p. 221
Bioassay for interleukin-16 (IL-16) p. 221
Bioassay for interleukin-17 (IL-17) p. 221
Bioassay for interleukin-18 (IL-18) p. 222
Bioassays for colony-stimulating factors--interleukin-3 (IL-3), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem-cell factor (SCF), thrombopoietin (TPO), and leukaemia inhibitory factor (LIF) p. 223
Introduction p. 223
Bioassay for leukaemia inhibitory factor (LIF) p. 227
Bioassay for thrombopoietin (TPO) p. 227
Bioassays for TGF-[beta] and related proteins p. 228
Bioassay for TGF-[beta] p. 228
Bone morphogenetic proteins p. 229
Bioassays for other cytokines p. 231
Bioassays for tumour necrosis factors p. 231
Bioassay for interferons (IFNs) p. 232
Bioassay for oncostatin-M p. 234
Analysis of results p. 235
Acknowledgements p. 238
References p. 238
List of suppliers p. 241
Index p. 251
Cytokine cell biology : a practical approach /
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