Measurement of Intracellular ROS Levels

The DCFDA/H2DCFDA-Cellular ROS Assay Kit (Abcam, Cambridge, UK; #ab113851) was used to measure the intracellular ROS levels, as previously described [15 (link)]. Briefly, 20,000 cells were plated in a 96-well microplate. After overnight culture, these cells were washed with the 1x buffer solution supplied in the kit, and treated with 20 μM DCFDA solution in 1x buffer for 45 min. After washing twice with PBS, the cells were placed in the culture medium containing compounds, and the DCF signals were measured at various times using an ARVO MX plate reader (PerkinElmer Inc.) or a SPARK plate reader (Tecan Inc.), with an excitation/emission wavelength of 485/535 nm. The values obtained from the blank condition (no cells) were subtracted from the DCF signal values.