![Fig. 3. Transduction of apoptosis-inducing proteins into CLL B cells by electroporation. / (A) CLL B cells from fludarabine-sensitive patients were incubated with BSA (panels Ai-Aii), caspase-8 (12 nM) (panel Aiii), or cytochrome c (60 μM) (panel Aiv), in the presence of PE-BSA (4 μg/mL) for 10 minutes on ice and were then left untreated (panel Ai) or electroporated with the use of 1125 V/cm and 900 μF. The percentage of cells that took up PE-BSA was determined by FACS analysis (FL-2 channel versus forward scatter [FSC]). R1 represents the selected gate containing the successfully transduced cells (PE+ population). (B) After electroporation, the cells transduced with BSA (panels Bi, Biv), caspase-8 (panels Bii, Bv), and cytochrome c (panels Biii, Bvi) (described in “Immunoblot analysis”) were cultured for 1 hour (panels Bi-Biii) or 3 hours (panels Biv-Bvi) at 37°C before being incubated with the fluorogenic caspase substrate CaspaTag for an additional hour at 37°C. Then, 7-AAD was added to the cells, and the percentage of CaspaTag–positive (FL-1 channel) and 7-AAD+(FL-3 channel) cells contained in the R1 gate (PE+) was determined by FACS. (C) To analyze the status of the 2 apoptosis pathways (the death receptor–mediated [extrinsic] or the mitochondria-mediated [intrinsic] pathway), CLL B cells from another fludarabine-sensitive patient were analyzed with regard to caspase activation. BSA, caspase-8 (12 nM), or cytochrome c(60 μM) was transduced into CLL cells with PE-BSA (4 μg/mL), in the presence or absence of specific inhibitors of caspase-8 (CrmA) (10 μM) or caspase-9 (XIAP-BIR3) (20 nM), by electroporation (1125 V/cm and 900 μF). Panel (i) shows CLL B cells transduced with BSA-CTR (CTR), or caspase-8 (C8); (ii) shows caspase-8 (C8), and caspase-8 + XIAP-BIR3 (C8 + BIR3); (iii) shows caspase-8 (C8), and caspase-8 + CrmA (C8 + CrmA); (iv) shows BSA-CTR (CTR), and cytochrome c (Cytc); (v) shows cytochrome c (Cyt c), and cytochrome c + XIAP-BIR3 (Cyt c + BIR3); and (vi) shows cytochrome c (Cyt c), and cytochromec + CrmA (Cyt c + CrmA). Effector caspase activity was measured 1 hour after electroporation by means of the cell-permeable fluorogenic caspase-3 substrate CaspaTag, flow cytometric cell analysis, and gating on the viable cell population (7-AAD−) that had taken up protein (PE-BSA+). A representative FACS histogram is presented, showing CaspaTag fluorescence (y-axis) versus forward scatter (x-axis) for the gated 7-AAD−, PE-BSA+ population. (Data shown are representative of 3 independent experiments.)](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/100/8/10.1182_blood-2002-04-1174/4/h82023276003.jpeg?Expires=1724263744&Signature=G~-eMgELCcY7-tTBL5xtHFa57YWCDIyrH4GLH3xuGCxQdUPb76MbPCxbJ7rT1Z06NHolWYAzrLsXXN3apWqVSsqp1KE754bKZQBCmB3UsiRVrGYQMKvhfiR5VzRIY0LhupGkdSdC68Jsa5zS6K~zpnl695UkYdL1zfRXx4LK666~Jm0ouopfKpSpA37SR3mCTcnlpFQUnV~InSU9ilWUmPgRMhuOb3tW-eDC5IAkg7YbgFO5K20rGpuh4TWRjioVBw3pHA2QPp~XnJJy9kSZrDbA2g7zJvR4A5VBUfwLoJVQRAm4K1N7ft5gedVf70ddwnSTlhH2JhkU6I6PhdcOpw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Transduction of apoptosis-inducing proteins into CLL B cells by electroporation.
(A) CLL B cells from fludarabine-sensitive patients were incubated with BSA (panels Ai-Aii), caspase-8 (12 nM) (panel Aiii), or cytochrome c (60 μM) (panel Aiv), in the presence of PE-BSA (4 μg/mL) for 10 minutes on ice and were then left untreated (panel Ai) or electroporated with the use of 1125 V/cm and 900 μF. The percentage of cells that took up PE-BSA was determined by FACS analysis (FL-2 channel versus forward scatter [FSC]). R1 represents the selected gate containing the successfully transduced cells (PE+ population). (B) After electroporation, the cells transduced with BSA (panels Bi, Biv), caspase-8 (panels Bii, Bv), and cytochrome c (panels Biii, Bvi) (described in “Immunoblot analysis”) were cultured for 1 hour (panels Bi-Biii) or 3 hours (panels Biv-Bvi) at 37°C before being incubated with the fluorogenic caspase substrate CaspaTag for an additional hour at 37°C. Then, 7-AAD was added to the cells, and the percentage of CaspaTag–positive (FL-1 channel) and 7-AAD+(FL-3 channel) cells contained in the R1 gate (PE+) was determined by FACS. (C) To analyze the status of the 2 apoptosis pathways (the death receptor–mediated [extrinsic] or the mitochondria-mediated [intrinsic] pathway), CLL B cells from another fludarabine-sensitive patient were analyzed with regard to caspase activation. BSA, caspase-8 (12 nM), or cytochrome c(60 μM) was transduced into CLL cells with PE-BSA (4 μg/mL), in the presence or absence of specific inhibitors of caspase-8 (CrmA) (10 μM) or caspase-9 (XIAP-BIR3) (20 nM), by electroporation (1125 V/cm and 900 μF). Panel (i) shows CLL B cells transduced with BSA-CTR (CTR), or caspase-8 (C8); (ii) shows caspase-8 (C8), and caspase-8 + XIAP-BIR3 (C8 + BIR3); (iii) shows caspase-8 (C8), and caspase-8 + CrmA (C8 + CrmA); (iv) shows BSA-CTR (CTR), and cytochrome c (Cytc); (v) shows cytochrome c (Cyt c), and cytochrome c + XIAP-BIR3 (Cyt c + BIR3); and (vi) shows cytochrome c (Cyt c), and cytochromec + CrmA (Cyt c + CrmA). Effector caspase activity was measured 1 hour after electroporation by means of the cell-permeable fluorogenic caspase-3 substrate CaspaTag, flow cytometric cell analysis, and gating on the viable cell population (7-AAD−) that had taken up protein (PE-BSA+). A representative FACS histogram is presented, showing CaspaTag fluorescence (y-axis) versus forward scatter (x-axis) for the gated 7-AAD−, PE-BSA+ population. (Data shown are representative of 3 independent experiments.)
