GSH Measurement

Levels of total reduced glutathione (GSH) were measured using a Glutathione (GSSH/GSH) detection kit from Enzo Life Sciences (Farmingdale, NY). The enzymatic assay is based on the interaction of the sulfhydryl group of GSH on DTNB (5,5′-dithiobis-2-nitrobenzoic acid, Ellman’s reagent) producing a yellow colored 5-thio-2-nitrobenzoic acid (TNB) that absorbs at 405 nm. The rate of TNB production directly correlates to the concentration of glutathione. Approximately 5×10⁶ MOLM-14, THP-1 and AML20 cells were exposed to vehicle, ATO (IC₅₀), DCA (IC₃₀) or their combination for 24 hours. As per the manual, after cells were washed in PBS, they were homogenized in 5% (w/v) metaphosphoric acid and frozen/thawed twice. The suspension was spun at 12,000 g for 5 minutes at +4^oC and the supernatant was frozen at −80^oC. After running the assay as directed by the kit manual, the absorbance was measured using Synergy HT, Multidetection Microplate Reader (BioTek) at 405 nm. GSH were measured using Graphpad Synergy software and its concentration was calculated in micromole per liter in cell lysate.