*4.4. Determination of Intracellular ROS Production*

The intracellular accumulation of ROS was determined by flow cytometry after staining with the fluorescent probe DCFH-DA (10 μM, 2 ,7 -dichlorodihydro-fluorescein diacetate; Molecular Probes, Invitrogen, Milan, Italy). DCFH-DA was deacetylated in cells by esterase to a nonfluorescent compound DCFH, which remains trapped within the cell and is cleaved and oxidized by ROS in the presence of endogenous peroxidase to a highly fluorescent compound DCF (2 ,7 -dichlorofluorescein). Cells were incubated with 10 μM DCFH-DA for 30 min at 37 ◦C, washed, and resuspended in PBS. ROS levels were monitored using a BD AccuriTM C6 (BD Biosciences).
