5 sulfo salicylic acid dehydrate

GSH and GSSG intracellular contents were detected by using a colourimetric assay (ThermoFisher Scientific—Life Technologies Corp, Carlsband, CA, USA). The analyses were performed according to the manufacturer’s instructions after treatments with azoles at 3 h of incubation; in brief, cell lysates were prepared in ice-cold 5% 5-sulfo-salicylic acid dehydrate (Sigma-Aldrich, St. Louis, MO, USA), and subsequently resuspended in the supplied diluent before adding them into a 96-well microplate. After promptly adding substrate, the reaction with the free thiol group of GSH initiates, producing a highly coloured product. The relative absorbance was detected at 405 nm. Assays were performed at least in triplicate for the GSH and the GSSG content detection. To determine oxidised glutathione content, 2-vinylpyridine (2-VP) (Sigma-Aldrich, St. Louis, MO, USA) was added to block free GSH or other thiols contained in each sample; the specific concentration of the GSSG was determined by performing a 2-VP-treated samples standard curve.

The intracellular contents of GSH and GSSG were detected with a colorimetric assay (Thermo Fisher Scientific; Life Technologies Corp, Carlsbad, CA, USA). After 3 h of incubation with azoles, the analyses were performed on cell lysates in line with the manufacturer’s instruction; in brief, cell lysates were added into a 96-well microplate (Corning Inc., New York, NY, USA), earlier prepared in an ice-cold 5% solution of 5-sulfo-salicylic acid dehydrate (Sigma-Aldrich, St. Louis, MO, USA), and subsequently diluted in the supplied buffer. Once the specific substrate had been added, the reaction started producing a colored product whose absorbance was detected at 405 nm by the Infinite M Plex, multimode (TECAN, Salzburg, Austria, Europe). The analyses were replicated at least three times per assay. 2-vinylpyridine (2-VP) (Sigma-Aldrich, St. Louis, MO, USA) was used to block free thiols and GSH, when the content of GSSG (the oxidized form of GSH) was analyzed; the GSSG content was then determined by comparing a 2-VP standard curve.