Facs software version 8

ROS and cell viability were quantified by flow cytometry using the fluorescence dye 5-(and-6)-chloromethyl 2’,7’- dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) (Life Technologies, Oregon, USA) and Ghost Dye (Tonbo Biosciences, San Diego, CA), respectively. The treated cells were thoroughly washed with PBS and resuspended in 1 mL of PBS containing 2% FBS supplemented with 2–5 μL of CM-H₂DCFDA and 1 μL of the Ghost Dye. The cells were then incubated at room temperature in the dark for 30 minutes and subsequently washed and resuspended in 300 μL of PBS containing 2% FBS. Dichlorodihydrofluorescein (DCF) emission at 525 ± 20 nm, which is proportional to the ROS generated in the cells, and emission of Ghost Dye at 780 nm, which is proportional to the cell viability, were detected by flow cytometry (BD Biosciences, San Jose, CA). The data were analyzed by using the BD FACS software version 8.