Intracellular ROS measurement

ROS production was quantified by the DCFH-DA method. The cell-permeant H₂DCFDA fluorescent probe is commonly used to detect cellular production of ROS. After treatment, the cells were loaded with 5 μM H₂DCFDA for 30 min at 37 °C, followed by washing three times with Hank’s balanced salt solution (HBSS). The fluorescence intensity (excitation = 485 nm; emission = 535 nm) was measured using an EnSpire® Multimode Plate Reader (Perkin-Elmer) and the photographs were obtained using an Axio Observer A1 fluorescence microscope (Carl Zeiss, Jena, Germany). Serving as the positive control, cells were treated with a well-known stress inducer, H₂O₂ at concentration of 1 mM [18]. Data are expressed as the percentage of fluorescence intensity of treated cells relative to untreated control.