Figure 2. Enhanced ROS, lipid peroxidation and externalization of phosphatidyl serine (PS) in RBC_VL after sensitization with anti-9-O-AcSGP IgG_VL.

A. Induction of ROS in sensitized RBC_VL. Prior to sensitization with anti-9-O-AcSGP IgG_VL or anti-9-O-AcSGP IgG_NHS RBC_VL and RBC_N were incubated with the hydroperoxide indicator H₂DCF-DA in PBS for 30 min at 37°C. Then the antibodies were added, the cell incubated as before and ROS generation determined by fluorimetry. As controls, ROS generation was determined after prior treatment of the RBC with N-acetyl cysteine, a quencher of hydroperoxides. The results are expressed as means ± S.D. (n = 5) of fold increases in comparison to the fluorescence levels detected with untreated erythrocytes. B. Increased lipid peroxidation in sensitized RBC_VL. Lipid peroxidation of erythrocyte membrane was detected and quantified with thiobarbituric acid (TBA) and the TBA-reactive species (TBA-RS) measured with a spectrophotometer at 532 nm. The results are mean ± S.D of fold increase in comparison with unsensitized RBC of five independent measurments. C–D. Enhanced externalization of phosphatidylserine on sensitized RBC_VL. Sensitized or unsensitized erythrocytes were incubated in annexin-V binding buffer with FITC-annexin-V and analyzed by flow cytometry. A23187-treated RBC in the presence of Ca²⁺ served as positive control. The results are shown as representative histogram of three independent experiments (C) and as comparative flow-cytometric analysis (D).