Determination of reactive oxygen species and reduced glutathione

Intracellular reactive oxygen species (ROS) levels were measured and quantified by DCFH-DA. Briefly, cells were treated for 48 h and incubated with DCFH-DA. DCFH-DA was deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’-dichlorofluorescein (DCF). Fluorescence emission intensity was measured using a microtiter plate reader (16). Intracellular glutathione (GSH) levels were measured using GSH assay kit (Sigma-Aldrich, USA) and according to manufacturer instructions (16).