Fig. 3.

Effect of nitrones on H₂O₂-induced ROS generation in BAEC. (A) BAEC were treated with 100 µM H₂O₂ alone at various time points (0–24 h) in the absence or presence of nitrone (0–50 µM). After treatment, cells were incubated with 25 µM of DCFH-DA at 37 °C for 30 min in the absence of light and ROS generation was then assessed using a FACS Calibur flow cytometer (BD) with excitation at 488 and emission at 535 nm. (Inset), Mean fluorescence intensity (MFI) form the histogram analysis of all three sets such as H₂O₂-alone (various time intervals), PBN + H₂O₂ and DMPO + H₂O₂ were presented as mean ± SEM, *p < 0.05 vs. control (untreated cells), and #p < 0.05 vs. H₂O₂-treated cells, where n = 3. (B) Confocal images of BAEC treated with H₂O₂ (100 µM) alone and those preincubated with nitrone (0–50 µM). The right panel corresponds to the merged image of DCF-fluorescence and DAPI-fluorescence, bar represents 10 µm. (C) A plot of relative DCF- fluorescence obtained from the average of 100 cells. Data are represented as the mean ± SEM, *p < 0.05 vs. control (H₂O₂-untreated cells), and #p < 0.05 vs. H₂O₂-treated cells, where n = 3.