*4.6. Measurement of Basal Intracellular Reduced Glutathione Levels*

The total glutathione level (GSSG + GSH) in HT-29, HCT116, DU154, and PC-3 cells were assayed by the Glutathione Assay Kit (Sigma, Milano, Italy), according to manufacturer's instructions. The protein concentration (µg/mL) was quantified by the Qubit fluorometer (Invitrogen, Milan, Italy) and the Quant-IT Protein Assay Kit (Invitrogen, Milano, Italy). Calibration was performed by the application of a two-point standard curve, according to the manufacturer's instructions.

Briefly, reduced glutathione (GSH) reacts with 5,5<sup>0</sup> -dithiobis(2-nitrobenzoic acid) (DTNB) in a recycling assay and produces glutathione disulfide (GSSG) and the 1,3,5-trinitrobenzene (TNB) anion, which can be detected by absorbance. In turn, the enzyme glutathione reductase then reduces GSSG, which release GSH that can react with another DTNB molecule. Therefore, the rate of TNB production is measured rather than a single determination of how much DTNB react with GSH, as it is proportional to the initial amount of GSH [61]. The plate was read at 412 nm on a microplate reader Asys UV 340 (Biochrom, Cambridge, UK) and the amount of GSH was expressed in nmol/µg protein.
