The intracellular ROS level was determined using a ROS assay kit and dichloro-dihydro-fluorescein diacetate (DCFH-DA). According to the manufacturer's protocol, the cells were cultured in 96-well plates for 24 h at 37°C and were then washed twice with serum-free medium. Medium containing 10 µM DCFH-DA was added. The cells were incubated in a cell incubator for 20 min at 37°C. Light was avoided during procedures and incubation. After incubation, the cells were washed three times with serum-free medium, then observed and photographed using a fluorescence microscope (magnification, ×400; Olympus Corporation). The fluorescence intensity was quantitatively measured using ImageJ software (v1.51; National Institutes of Health).

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