2.9. Measurement of ROS levels

The conversion of non-fluorescent DCFH-DA to fluorescent dichlorofluorescein (DCF) was used to monitor the intracellular ROS production as described previously [36] with minor modifications. In brief, cells were seeded in 6-well culture plates. After treatment, the cells were washed with PBS and harvested by trypsinization. The cells were then stained with 10 μM DCFH-DA for 30 min at 37 °C in the dark. The cells were then washed with PBS and the fluorescence intensity was analyzed immediately using a Synergy HTX Multi-mode Microplate Reader (excitation at 488 nm, emission at 525 nm).

The detection of mitochondrial ROS was performed as described previously [37] with minor modifications. Cells were co-stained with MitoSOX Red and MitoTracker Green. After treatment, cells were stained with MitoSOX Red mitochondrial superoxide indicator (5 μM) diluted in DMEM with 10% FBS at 37 °C for 15 min, and washed with PBS for three times. Cells were then stained with MitoTracker Green (180 nM) diluted in FBS-free DMEM at 37 °C for 30 min. Cells were washed by PBS and the mitochondrial ROS production was evaluated using an Olympus BX53 fluorescence microscope (excitation at 480–550 nm, emission at 590 nm).