Time course of cytochrome c release on apoptosis induction.

Jurkat cells (1 × 10⁶) were incubated with medium or staurosporine (Stauro, 2.5 μM) and harvested after the indicated time points. (A) Release of cytochrome c was determined by immunoprecipitation of the medium fractions and subsequent immunoblotting. Heavy and light chains of anticytochrome c antibody and cytochrome c are indicated by open and closed arrowheads, respectively. The LDH activity in the supernatant of apoptotic cells represents the percentage of total LDH activity in each sample. The cytochrome c content in the supernatant indicates the absolute densitometric values. (B) Relative kinetics of release of cytochrome c and LDH activity. To visualize the relative quantities of cytochrome c and LDH activity in the culture medium, the absolute densitometric values of cytochrome c and the percentage of released LDH from Figure 1A were divided by the control values (0 hours). (C) Apoptosis induction by staurosporine was assessed by flow cytometry. The histograms show measurements of hypodiploid DNA formation. The percentage of apoptotic hypodiploid cells is shown on the right.