*4.9. Lactate Dehydrogenase Leakage Assay*

Lactate dehydrogenase (LDH) is an enzyme that is widely present in cytosol and catalyzes the conversion of lactate to pyruvic acid. If plasma membrane integrity is disrupted, the LDH leaks into culture media, increasing its extracellular level, and the amount of LDH release is proportional to the number of damaged cells [62]. The LDH leakage was evaluated by the LDH-Cytotoxicity Detection Kit (Roche Diagnostic, Indianapolis, USA), according to manufacturer's instructions. Briefly, 96-well plates were seeded with HT-29, HCT116, DU145, and PC-3 cell lines at a density of 2.0 × 10<sup>3</sup> , 1.5 × 10<sup>3</sup> , 5.0 × 10<sup>2</sup> , and 1.2 × 10<sup>3</sup> cells/100 µL culture medium, respectively. Twenty-four hours after the seeding, 100 µL of different concentrations (0.5, 1.0, 2.0, and 3.0 mg/mL) of GSH-NS B or GSH-NS D was added to the wells. The plates were then incubated for 24, 48, and 72 h, at 37 ◦C, in a humidified atmosphere of 5% CO<sup>2</sup> air. Cell-free culture media were then collected and incubated with the same volume of reaction mixture for 30 min. LDH activity was measured at 490 nm on a microplate reader Asys UV 340. The background control was obtained by measuring the LDH activity of the assay medium, the untreated control by measuring the LDH activity of untreated cells, and the positive control by measuring the maximum releasable LDH activity after the treatment with the lysis buffer. The LDH leakage percentage was calculated as follows: LDH leakage (%) = (experimental value−untreated control)/(positive control−untreated control) × 100, and is the mean of three independent wells.
