Intracellular ROS level detection by DCFH-DA assay

Intracellular ROS levels were measure by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), as previously reported (Pocasap et al., 2018). DCFH-DA is oxidized by intracellular ROS to 2′,7′-dichlorofluorescein (DCF)—a fluorescent compound detected by flow cytometer. HepG2 cells were treated with ITCs at different concentrations (10, 20, and 40 μM) for 12 h. Cells were then collected and incubated with DCFH-DA (10 μM) in the dark for 30 min at room temperature. The cells were subsequently washed with PBS and analyzed using the FACS analyzer (BD FACSCanto II). Intracellular ROS levels were calculated by BD FACSDiva software and expressed as a percentage of DCF intensity vis-à-vis the cell population in each treatment.