Apoptosis induction in Bo cells: effects of time and increased cytochrome c concentrations and competition by yeast cytochrome c. (a) (Upper panels) Bo cells were electroporated with control protein (FCS, C) or bovine cytochrome c at 80, 160, or 240 μg/mL external concentration as indicated and recultured with IL-3. (Lower panels) Cells were coelectroporated with 80 μg/mL bovine cytochrome c and 80, 160, or 240 μg/mL yeast (Y) cytochrome c. PI staining and flow cytometric analysis was performed after 2 hours. Note the appearance of pre-G1 staining (gate B) coincident with loss of G2/M staining with cytochrome c and lack of competition by yeast cytochrome c. Pre-G1 fractions, gate B: control, 8%; 80 μg/mL cytochrome c, 38%; 160 and 240 μg/mL cytochrome c, 39%. Gate (A and B): less than 8% in all. (b) Same as for (a) analyzed after 4 hours. Apoptotic fractions increase with time, but not with increasing concentrations of cytochrome c. Note the increase in cells with low amounts of PI staining (gate A, arrow) and progressive loss in G2/M staining throughout all cultures; gate (A and B) increases from 6% in (a) at 80 μg/mL to 17% in (b).