Index 255. B lymphocytes, 26 Bak, 96 basal condition preparation, See also apoptosis measurement

Transcription

1 Index accurate apoptosis measurement, 23. See also apoptosis measurement activated Bax staining by flow cytometry, See also mitochondrial membrane potential active caspase-3, 15 active caspase-3 detection flow cytometric detection of, activated caspase-3 direct staining detection, activated caspase-3 indirect staining detection, apoptosis induction aspects, 51 equipment, 50 reagents, immunoblotting for, 15, materials colorimetric assay, 15 fluorescein-labeled caspatag pan-caspase in situ assay kit, 16 immunoblotting, 15 immunocytochemistry for, 15 methods colorimetric assay, 17 fluorescein-labeled caspatag pan-caspase in situ assay kit, 19 immunoblotting, 18 immunocytochemistry for, activity (caspase), differential, See also caspase activity assays activity-based probes (ABP), 207. See also caspases apoptotic extracts, hypotonic extracts, 210 living cells, recombinant proteins, AI. See apoptotic index ALDH. See enzyme aldehyde dehydrogenase algorithmic analysis. See also apoptotic markers AQUA, 91 PLACE, 92 RESA, 91 7-aminoactinomycin D (7-AAD), AAD/FSC, 25 flow cytometry method and, 114, flow cytometry-based total cytotoxicity assay, 114 for apoptosis detection, 117 for in vitro apoptosis detection, 116 stain, 120, 123 total cytotoxicity assay, 114, 117, aminoluciferin, 163. See also proteasome assays annexin V, 23. See also mitochondrial membrane potential labeling, 24 staining, antibodies. See also apoptotic markers for cancer-related protein detection, 38 target antibody concentration, target, 91 anti-cancer agents. See also ChemoFx assays dilution and application of, 66 preparation, removal, anti-cancer mechanism, phenoxodiol and, 6 antigen cancer, 4 CA125, 4 CK-7, 36 lineage antigen (LAg), 23 loss, 23 retrieval, 90 apoptosis. See also apoptotic caspases; mitochondrial membrane potential accurate apoptosis measurement, 23 Bax activation aspects, 95 caspase inhibitors, caspase-3 activation and, caspase-3 assays and colorimetric assay, 15, 17 fluorescein-labeled caspatag pan-caspase in situ assay kit, 16, 19 immunoblotting, 15 16, 18 immunocytochemistry, CD95-induced, 227 chemoresistance and, 4 5 detection. See under apoptosis detection dose-dependent, epithelial ovarian cancer (EOC) and, 79 flow cytometry and, 26 27

2 254 Index flow cytometric detection of activated caspase-3, 47 genotoxic stress-induced, inducers, 49 induction and flow cytometric detection of activated caspase-3, 51 inhibitors, chemoresistance and, 4 5 measurement. See under apoptosis measurement peptide-based inhibitors, 110 quantification, reverse chemoresistance and, 1 TRAIL-induced, 221, 227 tumor cell, 109 apoptosis detection 7-AAD vital stain, 116 apoptosis induction in experimental cell population, 117 cell culture aspects, 117 cell-culture wash procedure, 118 FLICA probe for, 111 FAM-DEVD-FMK, 116 FAM-VAD-FMK, 116 FLICA reagent based culture, 118 in vitro, 116 flow cytometry method, , 119 fluorescence plate reader method, 120 in vitro assay, 109, 111 FLICA probe for, 116 using 96-well fluorescence plate reader formats, 115, using flow cytometry, 115, in vivo assay, 109, 111, 115, 117 assay control, assay procedure, 126 flow cytometry analysis, 127 fluorescence microscopy observation, 126 methods, intracellular caspase detection in adherent and suspension cells, 112 total cytotoxicity assay for, , AAD stain, 117 assay control aspects, 122 assay procedure aspects, 122 flow cytometry set up, 123 non-fluorescent reagents, 116 SR-VAD-FMK, 116 target cell stain CFSE, 116 total cytotoxicity calculation, 125 using membrane permeant FLICA technology, 109 apoptosis measurement. See also apoptotic caspases activation and activity materials equipment, 28 reagents, 28 methods, 28 AR calculation, 30 basal condition preparation, cell culture preparation, 29 cell suspension preparation, 29 cells after culture, acquisition of, 30 microbeads preparation, apoptotic cancer cells, 23 apoptotic caspases, 2 3 chemoresistance and, 4 5 phenoxodiol anti-cancer mechanism, 6 8 as chemosensitizer, 8 XIAP regulation, 5 6 apoptotic caspases activation and activity, 191 caspase titration, equipment for, 194 executioner caspases, , immunoblot analysis for, initiator caspases, labeling using ABP, apoptotic extracts, hypotonic extracts, 210 living cells, recombinant proteins, materials for, 194 measurements in extracts, 201 detergent, hypotonic, measurements of recombinant caspases in vitro, , methods, activity measurement, caspase labeling using ABP, reagents for, streptavidin blot for, 214 apoptotic cell fragment, 24 apoptotic extracts labeling cell culture aspects and, 208, 209 extract preparation aspects, 209 streptavidin for, 209 measurements in, 201 detergent, hypotonic,

3 Index 255 apoptotic index (AI), 23, 26 apoptotic markers, 83 algorithmic analysis AQUA, 91 PLACE, 92 RESA, 91 antigen retrieval, 90 automated image and analysis, 91 deparaffinization, 90 equipment, 89 fluorescent IHC, immunohistochemistry aspects of, 83 incubation, 91 materials, 89 methods, peroxidases blocking, 90 protein blocking, 90 reagents, 89 statistical analysis, 92 target antibody concentration, choice of, 91 identification, 90 tissue microarrays for, 83 advantage, 84 automated analysis, cohort size, 86 contruction, disadvantage, marker expression and clinical outcome, association between, 86 apoptotic rate (AR), 23, 26 27, 30 apoptotic whole cells, 24 AQUA. See Automated QUantitative Analysis AR. See apoptotic rate automated analysis apoptotic markers expression and, 91 TMAs, Automated QUantitative Analysis (AQUA), 86. See also apoptotic markers; Pixel Locale Assignment for Compartmentalization of Expression (PLACE); tissue microarrays; Rapid Exponential Subtraction Algorithm (RESA) AWB, 119 B lymphocytes, 26 Bak, 96 basal condition preparation, See also apoptosis measurement Bax activation, mitochondrial membrane potential and activated Bax staining by flow cytometry, annexin V staining, DiOC6 staining, equipment, 97 FL5.12 cell culture and IL-3 deprivation, 98 JC-1 staining, materials, 97 methods, reagents, 97 TMRM staining, Bcl-2 family, 96 biochemical basis for substrate selectivity, bioluminescent proteasome assays, well plate, 175 cellular, controls blank control, inhibitor control, no-treatment control, equipment for, 169 in vitro, 169, proteasome catalytic activities inhibition curves, Proteasome-Glo Cell-based standard assay, 175 Proteasome-Glo reagent preparation, 170 Proteasome-Glo standard assay, 172 Proteasome-Glo assay condition, 172 material for, 169 methods, 170 proteasome activity from cultured cells, controls and assay conditions, 174 multiplexing Proteasome-Glo Cell-based assay and caspase activity assay, proteasome inhibition aspects, Proteasome-Glo Cell-based reagent preparation, 174 BJAB (Human Burkitt s Lymphoma Line) culture, See also death-induced signaling complex (DISC) blank control, See also bioluminescent proteasome assays blot analysis apoptotic caspase activation and activity

4 256 Index immunoblot analysis, streptavidin blot analysis, 214 immunoblot. See immunoblotting western, 235, 236 Burkitt s lymphoma, 230, 231 CA125. See cancer antigen 125 CAD. See caspase-activated deoxyribonuclease Caenorhabditis elegans, 14 cancer anti-cancer mechanism, 6 apoptotic cancer cells, 23 chemotherapy, 57. See also ChemoFx assay ovarian. See ovarian cancer cancer antigen 125 (CA125), 4 cancer-related proteins detection materials antibodies and, 38 equipment, 37 reagents for immunocytochemistry, 38 reagents for tissue preparation, 38 methods cell lysis, 42 documentation, 42 immunostaining protocol for fresh-frozen sections in preparation for LCM, 40 LCM, 40, 41 sample preparation, 39 slides preparation, 39, 40 specimen collection and freezing, 38 XIAP and, 37 carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor of caspase. See FAM-VAD-FMK caspase activity. See also apoptotic caspase activation and activity; caspase activity assays and chemo-response, correlation between, 80 measurement, 193 in extracts, in vitro, , measurement in extracts, 201 detergent extract, hypotonic extract, measurement, in vitro, 197, caspase titration, executioner caspases, , initiator caspases, multiplex. See multiplex caspase activity caspase activity assays, 137 biochemical basis for substrate selectivity, cell-culture biology considerations, equipment, 142 fluorescent caspase activity assay considerations, 147 functional basis of differential activity, luminescent caspase activity assay considerations, 148 materials, 142 methods, 142 caspase activation, 143 caspase inhibition, 143 fluorescent caspase activity assays, luminescent caspase activity assays, 145 Proteasome-Glo Cell-based assay multiplexing, 176 reagents, 142 caspase induction time course, 154. See also multiplex caspase activity caspase inhibition caspase activity assays, 143 of apoptosis, caspase-2, 3 4, 7 caspase-3, 3 4, 5 activation, genotoxic stress-induced apoptosis and, activity assay, epithelial ovarian cancer (EOC) and, 79 colorimetric assay, 15, 17 detection flow cytometric. See flow cytometric detection of activated caspase-3 immunoblotting for, 15 16, 18 immunocytochemistry for, fluorescein-labeled caspatag pan-caspase in situ assay kit, 16, 19 caspase-3/7 assay, multiplex viability and, 159 caspase-6, 4 caspase-7, 4 5, 159 caspase-8, 2 3 active, 49 like proteins, 4 oligomerization, 4 caspase-9, 3, 14 caspase-activated deoxyribonuclease (CAD), 14 Caspase-Glo assay. See also epithelial ovarian cancer (EOC) for 96-well plate, 81 lysates, 81

5 Index 257 caspases, 2 3 activation, 143, See also apoptotic caspase activation and activity caspase-2, 141 caspase-6, 141 caspase-7, 141 caspase-8, 140 caspase-9, 140 chemoresistance and, 4 5 effectors, 79 executioner, 197, FLICAs. See fluorescent-labeled inhibitors of caspases (FLICA) for apoptosis detection intracellular caspase detection in adherent and suspension cells, 112 group I, 138 group II, 138 group III, 139 ICE/CED-3, 14 initiator, 79 labeling using activity-based probes (ABP), apoptotic extracts, hypotonic extracts, 210 living cells, recombinant proteins, mammalian, 14 phenoxodiol anti-cancer mechanism, 6 8 as chemosensitizer, 8 selectivity caspase-6, 139 caspase-8, 139 caspase-9, 139 titration, XIAP regulation, 5 6 caspatag pan-caspase in situ assay kit, fluorescein-labeled, 16, 19 catalytic activities, proteasome, 172 CCCP. See cyanide m-chlorophenylhydrazone CD19 + lymphocytes, 25 CD19-APC, 25 CD95 DISC CD95-induced apoptosis, 227 cflip proteins and, 226 cell culture, 30 apoptosis measurement and, 29 apoptotic extracts labeling and, caspase activity assays, mirna expression profiling, 187 multiplex caspase activity, cell enumeration, 23 cell fragment, apoptotic, 24 cell fragmentation, 23 cell lysis, 42, 80 cell mediated cytotoxicity (CMC), 111, 113 cell suspension preparation, 29. See also apoptosis measurement cellular bioluminescent proteasome assay, See also bioluminescent proteasome assays cellular FLICE-like inhibitory protein (cflip), 221 CD95 DISC and, 226 cflip L, cflip S, CEZ. See chloroethylaziridine cflip. See cellular FLICE-like inhibitory protein CFSE. See succinimidyl ester ChemoFx assays, 58 chemosensitivity and resistance assay (CSRA), 57 comparison with CSRA, 59 for ex vivo treatment, 57 inter-day drug stability, 73 intra-day drug stability, 74 intra-operator process variability, 73 materials assay, 60 equipment, 60 immunocytochemistry, 60 tissue culture, methods, accessioning, explanting, and initiating culture, anti-cancer agents preparation, anti-cancer agents, dilution and application of, 66 anti-cancer agents, removal of, cultures examination, 62 cultures, trypsinization and counting, 63 flask actions, 62 fluorescence staining of cultures, immunocytochemistry, 64 plating cultures, results interpretation, staining and automated cell counting, reproducibility of, 72 sequential events timing, sensitivity to, 74

6 258 Index chemoresistance, 6 apoptosis inhibitors and, 4 5 apoptotic cascade and, 4 reverse, 1 XIAP regulation and, 5 chemoresistant EOC cells, 6 chemo-response and caspase activity, correlation between, 80. See also epithelial ovarian cancer (EOC) chemoresponse, ex vivo, 57 chemosensitivity and resistance assay (CSRA), See also ChemoFx assays chemosensitizer, phenoxodiol as, 8 chemotherapy, 57. See also ChemoFx assays chloroethylaziridine (CEZ), CK-7 antigen, 36 clearing process, paraffin-embedded tissue sections, 249 CMC. See cell mediated cytotoxicity cohort size tissue microarray, 83, 86 collection of specimen, cancer-related protein detection and, 38 colorimetric assay, active caspase-3, 15, 17 cryostat tissue sections, , 246. See also tissue sectioning flash freezing in, 249 isopentane and dry ice, 248 isopentane and dry ice slurry, 248 isopentane and liquid nitrogen, 248 CSRA. See chemosensitivity and resistance assay cyanide m-chlorophenylhydrazone (CCCP), 102 cytochrome C, 49, 95 cytotoxicity assays, 109 flow cytometry-based total, for apoptosis detection, 116, multiplex caspase activity and, caspase induction time course method, dose-dependent apoptosis in a cell-culture model method, equipment, 153 materials, 153 methods, multiplex viability and caspase-3/7 assay method, 159 reagents, 154 total. See total cytotoxicity assays death domain (DD), 221. See also TNF-related apoptosis-inducing ligand (TRAIL) death effector domain (DED), 225 death-induced signaling complex (DISC), 2, 4, 48 49, 221, 228. See also TNF-related apoptosis-inducing ligand (TRAIL) CD95 DISC analyses, 226 cell culture aspects BJAB, HCC, 230 equipment, 228 flow cytometric determination of receptor expression, immunoblot analysis for, 236 immunoprecipitation, lysis and, 234 SDS PAGE for, 235 materials, 228 methods, 230 reagents, 229 staining aspects, TRAIL, 225 western blot analysis for, dehydration, paraffin-embedded tissue sections, 249 depolarization, membrane, detergent extract. See also hypotonic extract, activity measurement, 205 cell cultures aspects, 203 preparation, diethyl pyrocarbonate (DEPC), 242. See also laser microdissection (LMD) DiOC6 staining, See also mitochondrial membrane potential DISC. See death-induced signaling complex DNA microarray technology, 184 dose-dependent apoptosis in a cell-culture model, See also multiplex caspase activity dot blot. See also immunoblotting mirna expression profiling and, 183 nylon membrane array, mirna preparation from, down-regulation, XIAP, 7 drug stability. See also ChemoFx assays inter-day, 73 intra-day, 74 dry ice, 248 effectors caspases, 79. See also initiator caspases epithelial ovarian cancer (EOC), 3, 5, 79 caspase activity and chemo-response, correlation between, 80 Caspase-Glo assay for 96-well plate, 81

7 Index 259 Caspase-Glo assay for lysates, 81 cell lysis, 80 equipment for, 80 reagents for, 80 tissue lysis, caspase-3 activity assay and, 79 in vitro chemo-response, 79 chemoresistant, 6 7 ex vivo chemoresponse, 57 treatment, ChemoFx assay, 57 executioner caspases, 198. See also initiator caspases activation by Granzyme B, 207 activation by initiator caspases, FADD. See Fas-associated death domain FADD-like interleukin-1 converting enzyme (FLICE), 4 FAM-DEVD-FMK flow cytometry detection assay, 118 in vitro apoptosis detection, 116 probe apoptosis detection probe, 120 for in vitro apoptosis detection, 116 FAM-VAD-FMK, 19. See also SR-VAD-FMK apoptosis detection assay in vitro, 116 in vivo, 115, 117, 125 fluorescence plate reader analysis, 118, 121 in vivo apoptosis, 126 probe caspase detection, 121 in vitro apoptosis detection, 116 inhibitor, well fluorescence plate reader method, 118 Fas, 49 Fas-associated death domain (FADD), 48 49, 221, 225 FasL, 35 fixation, paraffin-embedded tissue sections, 249 FL5.12 cell culture, 98. See also mitochondrial membrane potential FLICA. See fluorescence-labeled inhibitors of caspases FLICE (FADD-like interleukin-1 converting enzyme), 4 FLICE-inhibitory proteins (FLIP), 4 flow cytometric detection of activated caspase-3, materials equipment, 50 reagents, methods activated caspase-3 direct staining detection, activated caspase-3 indirect staining detection, apoptosis induction aspects, 51 flow cytometry, 13, 23 7-AAD, 119 activated Bax staining by, See also mitochondrial membrane potential apoptosis detection by, 119 quantification and, apoptotic whole cells and cell fragments, based total cytotoxicity assay, AAD, 114 SR-VAD-FMK, 114 detection assay, 118 FAM-DEVD-FMK, 118 for DISC analysis, for in vitro apoptosis detection assay, 115, for in vivo apoptosis detection assay, 127 total cytotoxicity assay set-up and, 123 fluorescein-labeled caspatag pan-caspase in situ assay kit, 16, 19 fluorescence for in vivo apoptosis detection, 126 multiplex caspase activity, 151 staining of cultures, 64 fluorescence labeled inhibitors of caspases (FLICA), 14, 16, 19 for apoptosis detection, 109, 111, 118 in vitro, 109, 116 in vivo, 109, 115 intracellular caspase detection in adherent and suspension cells, 112 membrane permeant, 109 of caspases, 109 probe for in vitro apoptosis detection assay, 116 fluorescence plate reader analysis apoptosis detection, 120 FAM-VAD-FMK, 118, well, 115, fluorescent caspase activity assays, , 147. See also luminescent caspase activity assays

8 260 Index fluorescent IHC, apoptotic markers expression and deparaffinization, 90 incubation, 91 peroxidases blocking, 90 protein blocking, 90 target antibody concentration, choice of, 91 target identification, 90 fragmentation, apoptotic cells, freezing of specimen, cancer-related protein detection and, 38 FSC, 25 FSC/SSC, 26 functional basis of caspases differential activity, irradiation, 13 gel (apoptotic caspase activation and activity) preparation for immunoblot analysis, running for immunoblot analysis, 213 for streptavidin blot analysis, 214 gene expression, LMD for, 241 genotoxic stress-induced apoptosis, Granzyme B, executioner caspases activation by, 207 HCC culture, 230. See also death-induced signaling complex (DISC) hematoxylin and eosin (H&E) tissues, 36 homogeneous proteasome assays. See bioluminescent proteasome assays HRP, 209. See also activity-based probes (ABP) HTB77 cell line, 72. See also ChemoFx assays hybridization, mirna, 183, hypotonic extract. See also detergent extract activity measurement, 203 cell culture aspects, 202 labeling, 210 preparation, 203 IAP. See inhibitors of apoptosis proteins ICAD/DFF45, 14 ICE/CED-3, 14. See also caspases IHC. See immunohistochemistry IL-3 deprivation, 98. See also mitochondrial membrane potential image analysis, automated, 91. See also apoptotic markers imaging, tumor, 109 immunoblotting. See also streptavidin blot analysis DISC-associated proteins, 236 for active caspase-3 detection, 15, 16, 18 for apoptotic caspase activation and activity, 211, 214 gel preparation, 212 running gels, 213 sample preparation, 213 transfer aspects, 213 immunocytochemistry. See also immunohistochemistry(ihc) cancer-related proteins, 38 ChemoFx assay, 60, 64 for active caspase-3 detection, immunofluorescence apoptotic microarrays and, 83 flow cytometric detection of activated caspase-3 and, 47 staining, 47 immunohistochemistry(ihc), 13, 36 apoptotic markers, 83 fluorescent (for apoptotic markers expression) antigen retrieval, 90 deparaffinization, 90 incubation, 91 peroxidases blocking, 90 protein blocking, 90 target antibody concentration, choice of, 91 target identification, 90 tissue microarray, 84 immunoprecipitation, 221 DISC, lysis and immunoprecipitation, 234 SDS PAGE for, 235 TRAIL DISC, 225 immunostaining, 40 in vitro apoptosis, 109, 111. See also in vivo apoptosis in vitro apoptosis detection 7-AAD stain, 116 FAM-DEVD-FMK stain, 116 FAM-VAD-FMK stain, 116 FLICA probe for FAM-DEVD-FMK, 116 FAM-VAD-FMK, 116 in vitro, 116 in vitro apoptosis detection assays. See also in vivo apoptosis detection assays FLICA probe for, 116

9 Index 261 non-fluorescent reagents or assay components for, 115 using 96-well fluorescence plate reader formats, 115, using flow cytometry, 115, in vitro chemo-response, epithelial ovarian cancer (EOC) and, 79 in vitro proteasome assays, 169, proteasome catalytic activities inhibition curves, Proteasome-Glo assay condition, 172 reagent preparation, 170 standard assay, 172 in vitro recombinant caspases, 197 caspase titration, executioner, , initiator, in vivo apoptosis, 109, 111. See also in vitro apoptosis FAM-VAD-FMK stain, 126 SR-VAD-FMK stain, 126 in vivo apoptosis detection assay, 115, 117. See also in vitro apoptosis detection assays FAM-VAD-FMK, 115, 117 methods, 125 assay controls, assay procedure, 126 flow cytometry analysis, 127 fluorescence microscopy observation, 126 SR-VAD-FMK, 115, 117 incubation, apoptotic markers expression and, 91 inhibition in vitro proteasome assays, 172 proteasome in cultured cells, inhibitor control, See also bioluminescent proteasome assays inhibitors of apoptosis proteins (IAP), 4 inhibitors, apoptosis, 4 5 initiator caspases, 79, 198, See also executioner caspases isopentane, 248 JC-1 staining, See also mitochondrial membrane potential labeling caspases using activity-based probes, apoptotic extracts, hypotonic extracts, 210 living cells, recombinant proteins, RNA, 187 LAg. See lineage antigen laser capture microdissection (LCM), 35 36, 241. See also laser microdissection (LMD) cancer-related proteins detection, 41 immunostaining protocol for, 40 pure cancer cells, obtaining of, 41 immunostaining protocol for fresh-frozen sections of cancer-related proteins in preparation for, 40 laser microdissection (LMD) cells collection aspects, 251 extracted RNA, quality of, 252 for gene expression, 241 RNase activity, 241, 245 RNase activitiy supressing aspects, 242, 245, 248 tissue sectioning aspects, 243 cryostat tissue sections, , 246, 248 paraffin-embedded tissue sections, , 249 section staining aspects, 245, 247, 250 tissue time aspects, 251 LCM. See laser capture microdissection lineage antigen (LAg), 23 24, liquid nitrogen, living cells, labeling in, cell culture aspects, 209 extract preparation aspects, 210 LMD. See laser microdissection luciferase, 163. See also proteasome assay luminescence, multiplex caspase activity, 151 luminescent caspase activity assays, 148, 145. See also fluorescent caspase activity assays lymphocytes B lymphocytes, 26 CD19 +,25 lysates, Caspase-Glo assay for, 81 lysis DISC immunoprecipitation and, 234 cell, 42. See also cancer-related proteins detection mammalian caspases, 14 markers, apoptotic, 83 algorithmic analysis AQUA, 91 PLACE, 92 RESA, 91

10 262 Index antigen retrieval, 90 automated image and analysis, 91 deparaffinization, 90 equipment, 89 fluorescent IHC, immunohistochemistry aspects of, 83 incubation, 91 materials, 89 methods, peroxidases blocking, 90 protein blocking, 90 reagents, 89 statistical analysis, 92 target antibody concentration, choice of, 91 identification, 90 tissue microarrays for, 83 advantage, 84 automated analysis, cohort size, 86 contruction, disadvantage, marker expression and clinical outcome, association between, 86 membrane depolarization, membrane permeant FLICA technology, 109 microarrays DNA, 184 tissue, 83. See also under tissue microarray (TMA) microbeads, 23, See also apoptosis measurement microdissection, laser capture, 35 micrornas (mirna), 183 enrichment, 187 extraction and enrichment from tissues and cell cultures, 187 hybridization, labeling, 187 mature, 184 microarray-based, 184 mirna expression profiling, 183 DNA microarray technology, 184 dot blot, 183 equipment, 185 hybridization aspects, 183 materials, 185 methods, 186 mirna extraction and enrichment from tissues and cell cultures, 187 preparation from oligo dot-blot nylon membrane array, oligodeoxynucleotide library, 183 phosphorimaging aspects, 183 reagents, 185 mitochondria membrane depolarization, MOMP, permeabilization, 95 PTP, 96 mitochondria outer membrane permeabilization (MOMP), mitochondrial membrane permeabilization, 95 mitochondrial membrane potential Bax activation and, 97 activated Bax staining by flow cytometry, annexin V staining, DiOC6 staining, equipment, 97 FL5.12 cell culture and IL-3 deprivation, 98 JC-1 staining, reagents, 97 TMRM staining, methods, transmembrane potential (MTP), staining aspects, mitochondrial transmembrane potential (MTP), MOMP. See mitochondria outer membrane permeabilization MTP. See mitochondrial transmembrane potential multiplex caspase activity, 155 cytotoxicity assays and, caspase induction time course method, dose-dependent apoptosis in a cell-culture model method, equipment, 153 materials, 153 methods, multiplex viability and caspase-3/7 assay method, 159 reagents, 154 fluorescence, 151 luminescence, 151 viability assays assays and, multiplex viability and caspase-3/7 assay, 159

11 Index 263 multiplexed caspase activity assays, 152 MyD88, 35 non-fluorescent reagents, total toxicity assay and, 116 no-treatment control, See also bioluminescent proteasome assays oligo dot-blot nylon membrane array, mirna preparation from, oligodeoxynucleotide library, mirna expression profiling and, 183 osteoprotegerin (OPG), 223, 225 ovarian cancer cancer-related proteins detection and, 35 epithelial (EOC), 3, 79 caspase activity and chemo-response, correlation between, Caspase-Glo assay for 96-well plate, 81 Caspase-Glo assay for lysates, 81 caspase-3 activity assay and, 79 cell lysis, 80 chemoresistant, 6 7 equipment for, 80 in vitro chemo-response, 79 reagents for, 80 tissue lysis, pakt in, 8 reverse chemoresistance and, 1 p45 XIAP, 7 pakt in ovarian cancer cells, 8 paraffin-embedded tissue sections, See also tissue sectioning dehydration and clearing process, 249 fixation, 249 PARP-1. See poly (ADP-ribose) polymerase peptide-based inhibitors, 110 permeability transition pore (PTP), 95 permeabilization, mitochondrial membrane, 95 peroxidases blocking, apoptotic markers expression and, 90 personalized medicine, 57 phenoxodiol, 1 anti-cancer mechanism, 6 as chemosensitizer, 8 phosphoproteins, 227 phosphorimaging, mirna expression profiling and, 183 Pixel Locale Assignment for Compartmentalization of Expression (PLACE), 92. See also Automated QUantitative Analysis (AQUA); Rapid Exponential Subtraction Algorithm (RESA) poly (ADP-ribose) polymerase (PARP-1), 13 14, 20 primary tissue culture, 57 pro-caspase-3, 14 pro-caspase-8, 49 pro-caspase-9, 49 profiling, mirna expression, 183 DNA microarray technology, 184 dot blot, 183 equipment, 185 hybridization aspects, 183 materials, 185 methods, 186 mirna extraction and enrichment from tissues and cell cultures, 187 preparation from oligo dot-blot nylon membrane array, oligodeoxynucleotide library, 183 phosphorimaging aspects, 183 reagents, 185 programmed cell death. See apoptosis proteasome (20S), 164 proteasome activity from cultured cells, 174 blank control, 174 controls and assay conditions, 174 inhibitor control, 174 no-treatment control, 174 proteasome inhibition aspects, Proteasome-Glo Cell-based assay multiplexing Proteasome-Glo Cell-based assay and caspase activity assay, reagent preparation, 174 test control, 175 proteasome assays 20S proteasome assay, 163 bioluminescent, 163, well plate, 175 controls and assay conditions, 174 in vitro, 169, methods, 170 multiplexing Proteasome-Glo Cell-based assay and caspase activity assay,

12 264 Index proteasome activity from cultured cells, proteasome catalytic activities inhibition curves, proteasome inhibition aspects, Proteasome-Glo assay, 170, 172, cellular bioluminescent, 168, 169 in vitro, 172 Proteasome-Glo, , ubiquitin proteasome pathway, 164 proteasome catalytic activities inhibition curves, Proteasome-Glo assays condition, 172 in vitro, 170, 172 reagent preparation standard assay, 172 Proteasome-Glo Cell-based assays caspase activity assay multiplexing, 176 proteasome activity from cultured cells, proteins blocking, apoptotic markers expression and, 90 DISC-associated, 235, 236 labeling, recombinant, proteins, cancer-related proteins detection materials antibodies and, 38 equipment, 37 reagents for immunocytochemistry, 38 reagents for tissue preparation, 38 methods cell lysis, 42 documentation, 42 immunostaining protocol for fresh-frozen sections in preparation for LCM, 40 LCM, 40, 41 sample preparation, 39 slides preparation, specimen collection and freezing, 38 XIAP and, 37 PTP. See permeability transition pore Rapid Exponential Subtraction Algorithm (RESA), 91. See also Automated QUantitative Analysis (AQUA); Pixel Locale Assignment for Compartmentalization of Expression (PLACE) reagents for flow cytometric detection of activated caspase-3, for reagents for immunocytochemistry, cancer-related protein detection, 38 recombinant caspases in vitro, activity measurement of, 197, caspase titration, executioner, , initiator, proteins, regulation sphingosine kinase (SK), 7 XIAP, 5, 6 RESA. See Rapid Exponential Subtraction Algorithm reverse chemoresistance, 1 ribonuclease (RNase) activity laser microdissection (LMD) and, 241, 245 supressing, 242, 245, 248 by lab equipment, 242 with diethyl pyrocarbonate (DEPC), 242 tissue section staining and, 245, 247, 250 tissue sectioning and, RNA labeling, 187 micro (mirna) DNA microarray technology, 184 dot blot, 183 equipment, 185 extraction and enrichment from tissues and cell cultures, 187 hybridization aspects, 183 materials, 185 methods, 186 oligodeoxynucleotide library, 183 phosphorimaging aspects, 183 preparation from oligo dot-blot nylon membrane array, reagents, 185 small molecular weight (smwrnas), 183 S1P. See sphingosine-1 phosphate sample preparation, laser microdissection (LMD) for, 241 SDS PAGE, DISC immunoprecipitation and, 235 sectioning, tissue. See also laser microdissection (LMD) cryostat tissue sections, , 246, 248

13 Index 265 paraffin-embedded tissue sections, , 249 section staining aspects, 245, 247, 250 selectivity. See also caspase activity assays caspases caspase-1, 138 caspase-2, 138 caspase-4, 138 caspase-5, 138 caspase-6, 139 caspase-8, 139 caspase-9, 139 substrate, small molecular weight RNAs (smwrnas), 183 sphingosine kinase (SK), 7 sphingosine-1 phosphate (S1P), 7 SR-VAD-FMK. See also FAM-VAD-FMK caspase detection probe, 124 flow cytometry-based total cytotoxicity assay, 114 for apoptosis detection, 116 in vivo apoptosis detection assay, 115, 117, total cytotoxicity assay, 114, 116, SSC, 26 SSC/FSC, 25 staining. See also ChemoFx assays; mitochondrial membrane potential activated Bax, activated caspase-3 direct, indirect, automated cell counting and, DiOC6, 98, 99 flow cytometry for DISC analysis and, fluorescence, 64 immunofluorescence, 47 JC-1, tissue microarray comparison with, 84 section, 245, 247, 250 TMRM, statistical analysis, apoptotic markers, 92 streptavidin. See also immunoblotting blot analysis for apoptotic caspase activation and activity, 214 for apoptotic extracts labeling and, 209, 214 stress-induced apoptosis, genotoxic, succinimidyl ester (CFSE), 116 stained target cells, 124 total cytotoxicity assay, 116, target antibody concentration, apoptotic markers expression and, 91 target cell stain, CFSE, 116. See also apoptosis detection terminal deoxynucleotide transferase-mediated dutp nick-end labeling (TUNEL), 20 test control, 175. See also bioluminescent proteasome assays tissue cultures mirna expression profiling, 187 primary, 57 tissue lysis, tissue microarray (TMA), 83 advantages, 84 automated analysis, cohort size, 86 comparison with staining, 84 construction, 84 disadvantage, for apoptotic markers expression algorithmic analysis, antigen retrieval, 90 AQUA algorithmic analysis, 91 automated image and analysis, 91 deparaffinization, 90 equipment, 89 fluorescent IHC, incubation, 91 materials, 89 methods, peroxidases blocking, 90 PLACE algorithmic analysis, 92 protein blocking, 90 reagents, 89 RESA algorithmic analysis, 91 statistical analysis, 92 target antibody concentration, choice of, 91 target identification, 90 immunohistochemistry, 84 large cohort, 83 marker expression and clinical outcome, association between, 86 tissue sectioning. See also laser microdissection (LMD) cryostat tissue sections, , 246, 248 paraffin-embedded tissue sections, , 249 section staining aspects, 245, 247, 250 titration, caspase, TMA. See tissue microarrays TMRE (ethyl ester), 99

14 266 Index TMRM staining, See also mitochondrial membrane potential TNF superfamily (TNFSF), 221 TNFR1. See tumor necrosis factor (TNFR1) TNF-related apoptosis-inducing ligand (TRAIL), 2, 49, 155, See also death-induced signaling complex (DISC) CD95 DISC analyses and, 226 cellular receptors bind to, 223 cflip proteins and, 226 DISC, 225 induced apoptosis, 221, 227 OPG and, 225 TRAIL-R1, 222, TRAIL-R2, TRAIL-R3, TRAIL-R4, TNFRSF. See tumor necrosis factor (TNF) receptor superfamily TNFSF. See TNF superfamily toluidine blue, 247, 250 total cytotoxicity assays 7-AAD, 117, CFSE, 116, for apoptosis detection, 116, methods performing assay control aspects, 122 assay procedure aspects, 122 flow cytometry set up, 123 total cytotoxicity calculation, 125 SR-VAD-FMK, 116, TRAIL. See TNF-related apoptosis-inducing ligand (TRAIL) trypsinization, 63, 70 tumor cells apoptosis, 109 TRAIL-resistant, 221 tumor imaging, 109 tumor necrosis factor (TNFR1), 2 tumor necrosis factor (TNF) receptor superfamily (TNFRSF), tumors, ovarian cancer, 36 TUNEL. See terminal deoxynucleotide transferase-mediated dutp nick-end labeling ubiquitin proteasome pathway, 164 viability assays and caspase-3/7 assay, 159 multiplex caspase activity and, well fluorescence plate reader analysis, 81 AWB and, 119 FAM-VAD-FMK and, 118 for in vitro apoptosis detection assay, 115, Proteasome-Glo cell-based standard assay, 175 western blot analysis, DISC-associated proteins, See also blot analysis whole cells, apoptotic, 24 XAF-1. See XIAP associated factor 1 X-linked inhibitor of apoptosis protein (XIAP), 1, 5, 35 cancer-related proteins detection and, 37 down-regulation, 7 p45, 7 regulation, 5, 6 XIAP associated factor 1 (XAF-1), 5 zymogenicity, 139