Measurement of cellular reactive oxygen species production

ROS production in HepG2 cells was examined using 2’,7’-dichlorofluorescein diacetate (DCFDA). Cells were cultured and treated as previously described in 2.4. Cells were washed thrice with ice-cold PBS then incubated with 10 mM of DCFDA in the dark for 30 min. A microplate reader was used to measure fluorescence at the emission of 530 nm and the excitation of 502 nm, and the intensity and area (%) of cellular ROS were scanned by confocal fluorescence microscopy (Axio observer A1, Carl Zeiss, Jena, Germany) using a 40 × objective.