Effect of Hcy on intracellular ROS production. Cells were cultured without serum for 24 h and then treated with Hcy (100, 500, 1000 μM) for another 24 h. The intracellular ROS level of cells was detected by fluorescence microscope and flow cytometry using the fluorescent probe DCFH-DA (10 μM, 30 min). H2O2 (100 μM, 30 min) treatment is a positive control. (A) The ROS production was detected by DCFH-DA staining. The images are representative of typical staining (400×; scale bar: 20 μM). Digital scans of DCHF-DA-stained cells were quantified using Image J software (B). Data are shown as means ± SEM of three independent experiments. (C) The ROS levels were detected by flow cytometry analysis. Data are represented as percentages of the control group from six independent experiments (*P < 0.05 vs. control).