Induction of apoptosis by cytochrome c in Bo cells. (a) DNA analysis by flow cytometry. Cells were electroporated with bovine cytochrome c (Sigma Ltd) at different concentrations or control protein (FCS) and examined by flow cytomery after 4 hours of incubation. (Top row) With IL-3; (bottom row) without IL-3. Apoptosis is detectable at 8 μg/mL cytochrome c (middle panels) and is significant at 80 μg/mL (left panels, arrow). Apoptotic fractions (gate a, arrow) were as follows: with IL-3: control, <1%; 8 μg/mL, 2%; 80 μg/mL, 11%; without IL-3: control, <1%; 8 μg/mL, 5%; 80 μg/mL, 20%. (b) Apoptosis identified by TUNEL-labeling and flow cytometry. Intact cells were gated by forward- and side-scatter profiles. Fluorescence gates (A, B, C, and D) were then set for all positive events determined by Fluorospheres (Coulter Inc). The percentage of TUNEL-positive cells is in brackets. All cells were incubated with IL-3 and were electroporated with 80 μg/mL cytochrome c or equivalent amounts of FCS (control cells). (A and B) Bo cells examined by flow cytometry after 2 hours. (A) Controls (4%); (B) with cytochrome c (42%); (C and D) B15 cells after 4 hours of incubation. (C) Controls (11%); (D) with cytochrome c (36%). (c) Uptake of cytochrome c by electroporated cells. Bo cells were electroporated as described above with 80 μg/mL cytochrome mixed with 2 μg/mL biotinylated cytochrome c labeled with FITC-streptavidin. Cells were washed and recultured for 1 hour without IL-3 before flow cytometric analysis. (Left) Control cells electroporated with unlabeled cytochrome c; (right) with labeled cytochrome c. Twenty-four percent of cells were labeled. Twenty-seven percent of cells were apoptotic by direct microscopy at 2 hours. In 3 experiments, 31% ± 5.5% were labeled by FITC-cytochrome c and 36% ± 6% were apoptotic after 2 hours. Controls showed 2% ± 1.4% apoptotic cells.