Fig. 4. The protective effect of heparin to eryptotic death. RBCs were incubated with the anti-TLR2 Ab or isotypic Ab for 1 h and probed with a secondary Ab conjugated with FITC to show the TLR2 on the red cell surface (a). RBCs were first pre-incubated with mouse monoclonal anti-human TLR2 or TLR4 blocking Abs (50 μg/mL) or control mouse IgG2a for 20 min at room temperature, and then treated with medium alone or EHs (80 μg/mL) at 37°C for 24 h in a Ca²⁺ HEPES solution (b). 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. Heparin (1 U/mL) was added to EHs (40 or 80 μg/mL) for 15 min at room temperature for pre-treatment before culture (c). For post-treatment, Heparin (1 U/mL) was added after EHs treatment at the time point as indicated (d). Subsequently, cells were incubated at 37°C for 3 h and doubly stained with annexin V-RFP and calcein/AM for 30 min. Results are mean ± SD (n=3), *p<0.05, **p<0.01 relative to control. #p<0.05, ##p<0.01, relative to the response of the same EHs concentration.