Preface......Page 6
Contents......Page 8
Contributors......Page 10
1 Introduction......Page 13
2.1 Preparation and Cultivation of Primary Microglial Cells......Page 14
2.3 Stimulation of Microglia with Aβ for Inflammasome-Associated Assays (e.g., IL1β ELISA)
......Page 15
3.1 Preparation and Cultivation of Primary Microglial Cells ( See Note 2)......Page 16
3.2.1 Preparation of Aβ Monomers......Page 17
3.3 Stimulation of Primary Microglia with Aβ for IL-1β ELISA
......Page 18
4 Notes......Page 19
References......Page 20
1 Introduction......Page 21
2.3 Analysis of Active Caspase-1 in Macrophages Using a Cleavable Fluorescent Substrate......Page 23
3.1 Preparation of IAPP Oligomers ( See Notes 1 and 4)......Page 24
3.3 Analysis of Active Caspase-1 in Macrophages Using a Cleavable Fluorescent Substrate......Page 25
3.5 ELISA Analysis of Mouse IL-18......Page 26
4 Notes......Page 27
References......Page 29
1 Introduction......Page 31
2.2 Reagents......Page 32
3.1 Quantification of Lysosomal Loss of Fluorescence by FACS......Page 34
3.2 Assessing Lysosomal Rupture and Translocation by Confocal Microscopy......Page 35
4 Notes......Page 36
References......Page 39
1 Introduction......Page 40
2.3 Quantitative PCR for IL-1β and IL-18......Page 42
2.5 Western Blot for Detection of the Pro-form and Cleaved Form of (Pro-)Caspase-1, (Pro-)IL-1β and (Pro-)IL-18......Page 43
3.1 Cell Stimulation Assays (Monocytes and Macrophages)......Page 44
3.4 ELISA for Uncleaved Pro-IL-1β and Cleaved Mature IL-1β and IL-18......Page 45
3.5 Western Blot for Detection of the Pro-form and Cleaved Form of (Pro-)Caspase-1, (Pro-)IL-1β, and (Pro-)IL-18......Page 46
4 Notes......Page 47
References......Page 49
1 Introduction......Page 51
2.1.2 Priming and Stimulation of BMDCs......Page 53
2.1.3 ELISA Analysis......Page 54
2.1.4 Western Blot Analysis......Page 55
2.1.5 Determination of ASC Oligomerization Using Confocal Bioimaging of Live THP-1 Cells......Page 56
3.1.1 Murine Bone-Marrow Derived Dendritic Cell Differentiation
......Page 57
3.1.3 ELISA Analysis of BMDC Supernatants......Page 59
3.1.4 Western Blot Analysis......Page 60
3.1.5 Determination of ASC-Activation Using Confocal Bio-imaging of Live THP-1 Cells......Page 62
3.2.1 BMDC Cultures......Page 63
3.2.3 LIVE/DEAD ® Aqua Staining......Page 64
4 Notes......Page 65
References......Page 72
Website Reference......Page 73
1 Introduction......Page 74
2.1 Differentiation and Long-Term Storage of Murine BMDMs......Page 77
2.4 Measurement of Pyroptosis and Cytokine Maturation......Page 78
2.5 Analysis of Caspase-1 Processing by Western Blotting......Page 79
3.1 Differentiation and Long-Term Storage of Murine BMDMs......Page 80
3.2 Thawing and Propagating BMDMs......Page 82
3.4 Measurement of Pyroptosis and Cytokine Maturation......Page 83
3.5 Analysis of Caspase-1 Processing by Western Blotting......Page 85
3.6 Visualization of Endogenous ASC Oligomers......Page 86
4 Notes......Page 87
References......Page 91
1 Introduction......Page 94
2 Materials......Page 96
3.2.2 LDH Assay, Noncommercial......Page 97
4 Notes......Page 98
References......Page 99
1 Introduction......Page 100
2.2 Selecting Suitable Clones: Crude Selection......Page 102
2.6 Imaging and Imaging Analysis......Page 103
3.2 Selecting Suitable Clones: Crude Selection......Page 104
3.3 Selecting Suitable Clones: Limiting Dilution......Page 105
3.6 Imaging and Imaging Analysis......Page 106
4 Notes......Page 107
References......Page 110
1 Introduction......Page 111
2.2.2 Trichloroacetate Precipitation [ 4 ]......Page 112
2.4 Immunoblotting Reagents......Page 113
3.1 Cell Stimulation......Page 114
3.2.1 Methanol/Choloroform Precipitation......Page 115
3.2.2 Trichloroacetate Precipitation......Page 116
3.3.2 Tris–Glycine-PAGE Electrophoresis......Page 117
3.4 Gel Transfer......Page 118
3.5 Incubation with Antibodies and Blot Exposure......Page 119
4 Notes......Page 121
References......Page 123
1 Introduction......Page 124
2.1 Inflammasome Activation......Page 127
2.2 Inflammasome Measurement......Page 129
3.1 Stimulation of BMDCs......Page 130
3.3 Western Blot......Page 132
4 Notes......Page 134
References......Page 141
1 Introduction......Page 143
2.3 Protein Purification......Page 145
2.4 In Vitro NLRP1 Inflammasome Assays......Page 146
3.1.1 Recombinant Virus Preparation......Page 147
3.1.3 Small-Scale Protein Expression......Page 148
3.2 Purification of Inflammasome Proteins......Page 149
3.3.1 Caspase-1 Activation Measurements......Page 151
3.3.2 In Vitro ATP Binding to NLRP1......Page 152
4 Notes......Page 153
References......Page 157
1 Introduction......Page 159
2.1 Measurement of Adenosine-5′- O -(3-thiotriphosphate) (ATPγS) Binding by NLR Proteins......Page 161
2.2 Photo-affinity Labeling of NLR Proteins with 8-Azido-ATP Analogs
......Page 162
3.1 Measurement of Adenosine-5′ - O -(3-thiotriphosphate) (ATPγS) Binding by NLR Proteins......Page 163
3.2 Photo-affinity Labeling of NLR Proteins with 8-Azido-ATP Analogs
......Page 167
3.3 Measurement of Steady State ATPase Activity of NLR Proteins......Page 168
3.3.2 Measuring ATPase Activity......Page 169
4 Notes......Page 170
References......Page 173
1 Introduction......Page 175
2.2 Tissue Culture and Transfection......Page 178
2.6 Western Blotting......Page 179
3.1 Cell Culture, Transfection, and Lysis......Page 180
3.3 Blue Native/SDS Two-Dimensional PAGE......Page 181
3.4 Protein Transfer and Western Blotting......Page 182
3.5 Stripping and Re-probing Western Blots......Page 183
4 Notes......Page 184
References......Page 188
1 Introduction......Page 190
2.4 Reagents......Page 192
3.1.3 Transfer of Microbial Communities by Oral Gavage......Page 193
3.2.1 Sampling and Processing of Fecal Material......Page 194
3.2.3 Comprehensive Sequencing Analysis of Microbial Communities......Page 195
4 Notes......Page 196
References......Page 198
1 Introduction......Page 200
2.2 Digestion of Fat......Page 203
3.1 Collection of Subcutaneous and Visceral Mouse Adipose Tissue Depots......Page 204
3.2 Digestion of Fat......Page 205
3.3 FACS Staining......Page 206
3.4 FACS Gating Strategy and Analysis......Page 207
3.5 Digestion and Analysis of Human Adipose Tissue......Page 209
4 Notes......Page 211
References......Page 213
1 Introduction......Page 215
2.1 Skin Assay......Page 216
3.1.1 Inducing Inflammation and Collecting the Inflamed Skin Tissue......Page 217
3.2 Peritoneal Assay......Page 219
4 Notes......Page 222
References......Page 225
INDEX......Page 226