Fig. 3. Role of caspase-3 in the EHs-mediated eryptosis. Freshly isolated human RBCs (1.5x10⁷/mL) were treated with medium alone, ionomycin (1 μM) or EHs at the concentration as indicated in the absence (a) or presence (b) of caspase-3 inhibitor (Ac-DEVD-CHO, 200 nM) at 37°C for 3 h in a Ca²⁺-containing (a,b) or Ca²⁺-free (c) buffer. After incubation, cells (1x10⁶/mL) were doubly stained with annexin-V-RFP and FAM-DEVD-FMK for 20 min and subjected to flow cytometric analysis. Results (% of sub-population of annexin-V^high/FAM-DEVD-FMK^high (a,b) or MFI (c)) are mean ± SD (n=3), *p<0.05, **p<0.01, ***p<0.001, relative to control. ##p<0.01, relative to the response of the same concentration of EHs.