Figure 8.

OS-induced annexin-V binding was calcium-independent. RBCs (0.5 × 10⁹ cells/mL) were incubated with indicated concentrations of t-BOOH, A23187, and SNC in HEPES buffer containing 2 mM Ca²⁺ or 2 mM EGTA for indicated times. Annexin-V (0.1 µg/mL, 15 min, 25 °C) was added to treated cells and analyzed by flow cytometry. (A) Representative annexin V/FSC dot plots of one out of six independent experiments for 24 h. Gate G1 corresponds to control cells; G2 to annexin-V-negative EVs; G3 to annexin-V-positive cells; G4, annexin-V-positive EVs. (B,C) Calculation of annexin-V-positive events in G3 and G4. Data in (B–D) are presented as the mean ± SD, in (B)—paired t-test; ** p < 0.001, compared to 3h in HEPES buffer with Ca²⁺; ## p < 0.001, compared to 3 h in HEPES buffer with EGTA; n.s.—not significant. In (C)—one-way ANOVA, Levene’s test < 0.05, Tamhane’s T2 post hoc; * p < 0.05, ** p < 0.001, compared to 3 h control; # p < 0.05, ## p < 0.001, compared to 24 h control.