Fig. 2. EHs increased [Ca²⁺]i and ROS in human RBCs. Freshly isolated human RBCs (1.5x10⁷/mL) were treated with medium alone, ionomycin (1 μM), H₂O₂ (0.03%) or EHs at the concentrations as indicated at 37°C for 3 h in the Ca²⁺-containing or -free HEPES buffer. After incubation, cells (1x10⁶/mL) were doubly stained with annexin-V-RFP and fluo-4/AM for 30 min (a), annexin-V-RFP and CM-H₂DCFDA (5 μM) for 20 min (c, d) or annexin-V-RFP and calcein/AM (1 μM) for 20 min (b) in the Ca²⁺-containing buffer for flow cytometric analysis. Results are mean ± SD (n=3), *p<0.05, **p<0.01, ***p<0.001 relative to control; #p<0.05, ##p<0.01, relative to the response of the same concentration in the Ca²⁺-free HEPES buffer.