Measurement of intracellular ROS

The level of intracellular reactive oxygen species was quantified using the Reactive Oxygen Species Assay Kit. DCFH-DA is oxidized by reactive oxygen species in viable cells to 2′,7′-dichlorofluorescein (DCF), which is highly fluorescent at 530 nm. The cells were washed 3 times with PBS. DCFH-DA, diluted to a final concentration of 10 μM, was added and incubated for 30 min at 37°C in the dark. After being washed 3 times with PBS, the relative levels of fluorescence were quantified using a flow cytometer (530 nm).