Figure 5. Flow cytometry analysis of plasma membranes with annexin V-FITC/PI double staining. Cells were incubated either in the absence of mitomycin C for 15 h and 24 h (control, A) or in the presence of 0.001 mg/ml of mitomycin C for 24 h (B) and 0.01 mg/ml mitomycin C for 15 h (C) and 24 h (D). Undamaged cells were stained with negative annexin V-FITC/PI (bottom left quadrant). After incubation with 0.001 mg/ml mitomycin C for 24 h, a significant number of apoptotic cells were stained with positive annexin V-FITC and negative PI (bottom right quadrant). Following the increasing of mitomycin C to 0.01 mg/ml, advanced apoptotic cells stained by positive annexin V-FITC and PI (upper right quadrant) were significantly augmented from 12%±3% of the control to 34%±3% after 15 h and 49%±4% after 24 h incubation. During advanced apoptosis stages, the cells were no longer viable. Data are presented as means±SD from triplicates and three independent experiments. Two other independent experiments produced similar results.