Determination of ROS

Cells were seeded in the 96-well tissue culture plates, incubated for 24 h, and then exposed to different treatments. ROS were determined by ROS Assay Kit with fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). As a nonpolar compound, DCFH-DA can cross the cell membrane, be deesterified intracellularly, and turn to 2′,7′-DCFH, which is nonfluorescent and cannot cross the cell membrane. In the presence of intracellular ROS, DCFH is quickly oxidized to highly fluorescent 2′,7′-dichlorofluorescein. This fluorescence can be used for quantifying intracellular ROS production by a fluorescence microplate reader with excitation and emission wavelengths at 488 nm and 525 nm, respectively.