*4.12. Reactive Oxygen Species Production Assay*

The production of intracellular reactive oxygen species (ROS) was measured by flow cytometry using dichlorofluorescein-diacetate (DCFH-DA) (Sigma, Milano, Italy) as the oxidation-sensitive probe. Briefly, after 1, 12, and 24 h cell exposure to the respective GSH-NS B or GSH-NS D IC<sup>1</sup> and IC<sup>50</sup>

at 24 h, HT-29, HCT116, DU145, and PC-3 cells were washed twice with PBS in six-well plates and incubated with 10 µM DCFH at 37 ◦C in the dark for 30 min. The cells were then washed with PBS, trypsinized, collected in 500 µL of PBS, and analyzed. ROS production was expressed as iMFI ratio, that is, the difference between the iMFI of treated and untreated cells over the iMFI of untreated cells (iMFI is the product of the frequency of ROS-producing cells and the median fluorescence intensity).
