C5-triggered Cyt C release and drop in mitochondrial ΔΨ_mis dependent on opening of the MPT pore. (A) 0.5 mg purified rat liver mitochondria was exposed to 150 μg/mL C5 in the presence or absence of CsA (10 μmol/L) for 30 minutes at 30°C. Mitochondria were then pelleted, and supernatants were subjected to SDS-PAGE and western blot analysis for Cyt C, as described in Materials and methods. (B) Mitochondria were treated with C5 in the presence or absence of CsA as above and stained with membrane potential–sensitive dye DiOC₆ (40 nmol/L) at 37°C for 30 minutes, washed, and analyzed by flow cytometry for ΔΨ_m.