Programmed Cell Death Part A

Contributors......Page 2
Preface......Page 9
Methods in Enzymology......Page 11
Analyzing Morphological and Ultrastructural Features in Cell Death......Page 37
Introduction......Page 38
Tunel......Page 41
Cell viability......Page 42
Chemical fixation......Page 43
Preparation of Hoechst......Page 44
Methodology......Page 45
Cytospin preparation......Page 46
Mitochondria......Page 47
Cell permeation......Page 48
Electron Microscopy......Page 49
Fixation......Page 50
Buffers......Page 53
Dehydration......Page 54
"En block" staining......Page 56
Embedding......Page 57
Thin section staining......Page 58
Embedding protocol......Page 59
Scanning Electron Microscopy (SEM)......Page 60
References......Page 61
Evaluation of Some Cell Death Features by Real Time Real Space Microscopy......Page 63
Introduction......Page 64
Single cell measurements of DeltaPsim......Page 65
Bax, Mitochondria, and Calcium Signaling......Page 67
Generation of GFP-Bax constructs and evaluation Bax movement......Page 69
Analysis of cell morphology by transmission electron microscopy......Page 70
Bax effect on calcium signaling......Page 72
Calcium Signaling and Neurodegenerative Processes......Page 76
Huntington's disease and animal models......Page 77
Use of real time measurements in brain slices from transgenic mice......Page 78
References......Page 82
Analysis of Apoptotic Pathways by Multiparametric Flow Cytometry: Application to HIV Infection......Page 87
Introduction......Page 88
Intracellular Expression of Bcl-2 Family Members and Relationship with Apoptosis......Page 90
Control samples required for analysis of Bcl-2 expression......Page 91
Recombinant CAD......Page 307
Discrimination of cell subsets expressing variable levels of Bcl-2......Page 92
Ex vivo analysis of Bcl-2 expression in primary lymphocytes: Correlation with their priming for apoptosis......Page 93
Cells......Page 94
Materials and buffer......Page 311
Materials and buffer......Page 95
A drop in DeltaPsim is part of the apoptotic process induced in primary lymphocytes from HIV-infected subjects......Page 96
Protein Identification by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry......Page 380
The in situ nick translation assay......Page 99
Analysis of DNA fragmentation with the ISNT assay in peripheral lymphocytes prone to premature cell death......Page 100
The terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling assay......Page 102
Analysis with the TUNEL assay of DNA fragmentation in peripheral lymphocytes prone to premature cell death......Page 103
Annexin-V staining and Ca2+ dependence......Page 104
Application of the annexin-V assay to the analysis of AICD......Page 105
The 7-AAD assay......Page 107
Detection with the 7-AAD assay of early and late apoptotic cells on primary cells prone to premature cell death analysis......Page 108
A Flow Cytometry Assay to Quantify Cell-Mediated Cytotoxicity......Page 110
The CFSE/7-AAD cytotoxicity assay......Page 111
Definition of appropriate conditions for CFSE staining......Page 113
Analysis of cell-mediated cytotoxicity by the CFSE/7-AAD assay......Page 114
Acknowledgments......Page 115
References......Page 116
Methods to Analyze the Palmitoylated CD95 High Molecular Weight Death-Inducing Signaling Complex......Page 119
Introduction......Page 120
Equipment......Page 121
Assay procedure......Page 221
Buffers......Page 123
Example protocol for U937 cells......Page 124
Solutions......Page 249
Ultracentrifugation......Page 125
Protein transfer and Western blot......Page 126
Protocol......Page 314
Association of CD95HI with Active Caspase-8 (Reverse DISC-IP)......Page 127
Method......Page 129
Assay for Noncell-Autonomous DNA Degradation in Macrophages......Page 315
Role of Palmitoylation for CD95 Signaling......Page 130
Preparation of enriched [3H]palmitic acid......Page 131
Purification of GzmA protein......Page 258
In vivo metabolic labeling, immunoprecipitation, and fluorography......Page 132
Conclusions......Page 133
Acknowledgment......Page 134
References......Page 135
Immunomagnetic Isolation of Tumor Necrosis Factor Receptosomes......Page 137
Introduction......Page 138
Compartmentalization of TNF-R1 Apoptosis Signaling......Page 139
Methods for Magnetic Labeling and Isolation of Subcellular Signaling Structures......Page 140
Caspase activation in THP-1 cell-free extract......Page 377
Domains, structure, and assembly......Page 142
Experimental Procedures......Page 144
Detection of cathepsin activity in living cells......Page 227
Checking the efficiency of labeling with magnetic nanobeads......Page 147
Purification of ASC occlusion bodies......Page 148
Washing......Page 151
GRP78/BiP induction......Page 437
Washing of the magnetic chamber, purity, and specificity of the magnetic fractions......Page 152
Western blotting of isolated TNF receptosome proteins......Page 154
Labeling and isolation of CD95 receptosomes......Page 156
Conclusions......Page 157
References......Page 158
Analyzing Lipid Raft Dynamics During Cell Apoptosis......Page 160
Introduction......Page 161
Coimmunoprecipitation......Page 163
Glucose uptake......Page 165
Assaying the oligomeric nature of the ASC pyroptosome by chemical cross-linking......Page 166
TLC Immunostaining......Page 167
Procedure......Page 168
Protocol......Page 169
Protocol......Page 317
Postembedding electron microscopy......Page 170
Embedding medium......Page 171
Postembedding procedure......Page 172
References......Page 174
Apoptosome Assembly......Page 176
Introduction......Page 177
Processing of procaspases (Western blotting)......Page 179
Function of apoptosome......Page 182
Measurement of caspase activation by fluorescence/confocal microscopy or flow cytometry in intact cells......Page 183
Dark Apoptosome for Dronc Activation......Page 184
Pathway, structure, and assembly......Page 185
Protocol for assembly and detection of CED-4 apoptosome......Page 186
Acknowledgments......Page 187
References......Page 214
Caspases: Determination of Their Activities in Apoptotic Cells......Page 192
Introduction......Page 193
Methods for Analysis of Caspase Processing, Activation, and Substrate Cleavage......Page 197
Protocol......Page 198
Set up and run gel electrophoresis......Page 199
Immunoblotting of proteins......Page 200
Immunodetection......Page 201
In situ TG2 activity assay......Page 203
Affinity labeling and synthetic caspase inhibitors......Page 205
Detection of caspase activity using synthetic substrates......Page 207
Detection of caspase activity using synthetic inhibitors......Page 208
Using synthetic caspase inhibitors......Page 210
PARP cleavage......Page 211
Cytokeratin 18 cleavage......Page 212
Conclusion......Page 213
Lysosomes in Apoptosis......Page 217
Isolation of Myeloid Precursor Cells from Mouse Bone Marrow Cells......Page 218
Staining for Acid Compartments/Vesicles......Page 220
Validation of Lysosomal Membrane Permeabilization......Page 222
Assay procedure......Page 223
Lysosomal Cathepsins......Page 224
Synthetic Cathepsin Substrates......Page 225
Reagents......Page 226
Cathepsin inhibitors......Page 228
Cleavage of Protein Substrates......Page 229
References......Page 230
More Than Two Sides of a Coin?......Page 234
Introduction......Page 235
Radiometric assay......Page 236
Lipid extraction and two-dimensional (2D) high-performance thin-layer chromatography (HPTLC) analy......Page 237
TG2 Knockout Mice as Model System......Page 238
Immunocytochemistry and Immunofluorescence Detection of TG2......Page 240
In Vitro Phagocytosis Assays......Page 241
Conclusions......Page 243
References......Page 244
Granzymes and Cell Death......Page 246
Introduction......Page 247
Mitochondrial Outer Membrane Permeabilization......Page 342
Growth of RNK-16 cells in rats......Page 248
Assays for Cell-Autonomous Apoptotic DNA Degradation......Page 312
Purification of rat perforin......Page 250
Protocol......Page 251
Hemoglobin release assay......Page 252
Glut1 trafficking......Page 476
Expression and Purification of Recombinant Granzyme B in a Baculovirus System......Page 254
Virus amplification......Page 255
Expression and Purification of Recombinant human Granzyme A in Escherichia coli......Page 256
Protocol......Page 318
Replication of plate colonies on IPTG-coated plate......Page 257
Cleavage assay......Page 259
Chromium release assay......Page 260
Annexin/propidium iodide (PI) staining assay......Page 261
References......Page 262
Investigation of the Proapoptotic Bcl-2 Family Member Bid on the Crossroad of the DNA Damage Response and Apoptosis......Page 264
Introduction......Page 265
Materials......Page 268
Overexpression of glycolytic genes to increase glucose metabolism......Page 269
Purification of Lin- Cells......Page 270
Culture of Lineage-Depleted, Sca1+ Bone Marrow Cells......Page 271
Chromosomal Breakage Assays......Page 272
Materials......Page 273
Data Analysis......Page 274
Subcellular Fractionation......Page 275
Methods......Page 276
Immunofluorescence Staining on Adherent Cells......Page 277
Detection of LC3 by Western blot and immunohistochemistry......Page 333
Isolation of virally transduced cells......Page 278
Cell culture......Page 279
Annexin V Staining......Page 280
Controls......Page 281
References......Page 282
Assembly, Purification, and Assay of the Activity of the ASC Pyroptosome......Page 284
Introduction......Page 285
Construction of the ASC-GFP retroviral expression construct......Page 287
Confocal time-lapse bioimaging of live THP-1 cells......Page 290
Biochemical Assay of ASC Pyroptosome Formation in Stimulated THP-1 or Primary Macrophages......Page 291
Assembly and purification of ASC pyroptosomes from ASC-GFP expressing THP-1 cells......Page 294
Assembly and purification of endogenous ASC pyroptosomes from THP-1 cells......Page 296
In Vitro Assembly and Purification of Recombinant ASC Pyroptosomes......Page 297
Expression of ASC in bacteria......Page 298
Assembly of ASC pyroptosomes from occlusion bodies......Page 299
Assay of the activity of the pyroptosomes......Page 300
References......Page 301
Nucleases in Programmed Cell Death......Page 304
Introduction......Page 305
Assays for CAD......Page 306
Methods used to detect autophagy in cells......Page 325
Reagents......Page 457
Protocol for quantitative CAD assay......Page 310
Nuclear DNA fragmentation assay......Page 443
Materials and buffer......Page 316
References......Page 445
Detection of Autophagy in Cell Death......Page 321
Structural: Electron Microscopic Recognition of Lysosomes, Autophagic Vesicles, and Autophagosomes......Page 324
Mitochondrial Inner Membrane Permeabilization......Page 326
Bcl-2 markers for glucose metabolism......Page 327
Specific Uptake of Molecules into Lysosomes and Detection of These Molecules by Fluorescence Microscopy or Confocal Microscopy to Evaluate Number, Size, and Location of Lysosomes......Page 328
Monodansylcadaverine......Page 331
Analysis of internalization mechanisms......Page 366
Localization or Differential Modification of Autophagy-Related Genes......Page 332
LC3-GFP transfection to visualize LC3 translocation in C. elegans......Page 334
Methods used to detect autophagy in C. elegans using RNAi and mutations......Page 335
References......Page 336
Methods for Distinguishing Apoptotic from Necrotic Cells and Measuring Their Clearance......Page 339
Introduction......Page 340
Differential interference contrast microscopy......Page 343
Combination of DIC and epifluorescence microscopy......Page 344
Analysis of cell morphology versus cell permeability by flow fluorocytometry (FACS)......Page 345
Analysis of surface changes during apoptosis and necrosis by flow fluorocytometry (FACS): PS exposure versus cell permeability......Page 350
Analysis of cellular markers......Page 353
Analysis of activation of caspases by Western blotting......Page 354
Analysis of caspase activity using fluorogenic substrates......Page 357
Analysis of Bid cleavage and cytochrome c release by Western blotting......Page 358
Analysis of supernatants from apoptotic and necrotic cells......Page 359
Analysis of caspases release by Western blot analysis......Page 360
Analysis of HMGB-1 release by western blotting......Page 361
Analysis of Cell-Cell Interactions......Page 362
In vitro phagocytosis assay by flow fluorocytometry......Page 363
Analysis of phagocyte surface changes by scanning electron microscopy......Page 364
Transmission electron microscopy in combination with horseradish peroxidase......Page 367
Fluorescence microscopy in combination with lucifer yellow......Page 368
Acknowledgments......Page 369
Two-Dimensional Gel-Based Analysis of the Demolition Phase of Apoptosis......Page 374
Introduction......Page 375
Cell-Free Extract......Page 376
First dimension: Isoelectric focusing......Page 379
Destaining and trypsin digestion of silver-stained protein spots......Page 381
Matrix-assisted laser desorption/ionization time of flight mass spectrometry......Page 382
Notes......Page 397
Acknowledgments......Page 384
Methods to Dissect Mitochondrial Membrane Permeabilization in the Course of Apoptosis......Page 386
Introduction......Page 387
Immunofluorescence detection of the release of intermembrane space proteins......Page 390
Cytofluorometric assessment of mitochondrial transmembrane potential......Page 393
Calcein acetoxymethyl ester-based assays......Page 395
Guidelines for a Precise Determination of Mitochondrial Membrane Permeabilization......Page 396
Acknowledgments......Page 404
References......Page 421
Oxidative Lipidomics of Programmed Cell Death......Page 406
Introduction......Page 407
Reagents......Page 408
In vivo exposures......Page 409
Identification of hydroxy- and hydroxy-hydroperoxy-CL molecular species formed during peroxidation of TLCL bycytochrome c/H2O2 in a model system......Page 410
Identification of CL-hydroperoxides formed in vivo......Page 413
Identification of PS-hydroperoxides in a model system containing cytochrome c/H2O2......Page 416
Identification of PS-hydroperoxides in vivo......Page 419
Identification and Characterization of Endoplasmic Reticulum Stress-Induced Apoptosis In Vivo......Page 425
The Endoplasmic Reticulum (ER) and the Unfolded Protein Response......Page 426
The PERK/eIF2alpha pathway......Page 428
The IRE1alpha pathway......Page 429
Caspases and BCL2 family proteins......Page 431
Experimental protocols used to analyze UPR pathways associated with apoptosis in mouse liver......Page 432
Phosphorylation of IRE1alpha and PERK......Page 433
Xbp1 mRNA splicing......Page 435
Phosphorylation of eIF2alpha......Page 438
Experimental protocols for identification and characterization of ER stress-inducible proapoptotic factors in the liver......Page 439
Quantitative real-time RT-PCR analysis of Atf4 and Chop mRNAs......Page 440
Immunohistochemical staining of CHOP in the liver......Page 441
Organelle Intermixing and Membrane Scrambling in Cell Death......Page 450
Introduction......Page 451
Methods Used to Evaluate Intermixing of Mitochondria with Other Organelles......Page 452
Reagents......Page 453
Deconvolution fluorescence microscopy......Page 455
Protocol......Page 456
Intermixing of mitochondria with endosomes and other organelles......Page 458
Protocol for isolating perinuclear mitochondria......Page 460
Dispersal of the Golgi apparatus and related membranes: Single and dual HPA staining......Page 461
Protocol for HPA staining combined with other fluorescent markers......Page 463
Protocol of CtxB staining of live cells......Page 464
Acknowledgments......Page 465
Mechanisms and Methods in Glucose Metabolism and Cell Death......Page 468
Metabolism and forms of cell death......Page 472
How to monitor glucose metabolism......Page 473
Reagents......Page 474
Protocol......Page 475
Protocol......Page 478
Mitochondrial potential......Page 479
Inhibition of glucose metabolism......Page 481
References......Page 483
Author Index......Page 487
Subject Index......Page 513