Measurement of intracellular ROS concentration using DCF-DA probe

The production of ROS was determined by measuring the oxidative conversion of cell-permeable DCF-DA into fluorescent dichlorofluorescein (DCF). Cells were incubated with DCFH-DA (10 μM) diluted by serum-free medium in the dark at 37°C for 45 min prior to termination of treatment. Fluorescent emission of each well at 525 nm with the excitation wavelength of 488 nm was measured by a spectrophotometer (Multiscan MK3, ThermoFisher, USA). The concentration of ROS was expressed either as the absolute value of fluorescent emission, or as fold change in respect to the control group.