2.4. Cell ROS Determination by DCFH-DA

Intracellular formation of ROS was determined via the fluorescent probe DCFH-DA. Cells were plated in 24-well plates, and YGDEY (10, 20, 50, and 100 μM) was added to the wells. After 2 h, cells were exposed to 0.75 M ethanol. DCFH-DA (10 μM, 200 μL) was added to each well. After reacting for 20 min at 37 °C in the dark, cells were washed with phosphate buffer saline (PBS). The formation of 2’,7’-dichlorofluorescein (DCF), due to the oxidation of DCFH-DA via intracellular ROS, was immediately examined using an inverted fluorescence microscope (Olympus Opticals, Tokyo, Japan).