3.10. Measuring Reactive Oxygen Species (ROS)

Intracellular ROS levels were detected using 2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (H2DCFDA; Thermo Fisher Scientific, Waltham, MA, USA). For this analysis, cells were incubated with DCFDA (1 μM) for 15 min at room temperature. Cells were also washed with cold PBS and resuspended in 0.5 mL of PBS supplemented with 1% fetal bovine serum. Intracellular fluorescence accumulation was analyzed using a flow cytometer (BD FACSVerse, BD Biosciences, San Diego, CA, USA).