Gsh detection assay kit

The relative GSH concentration in cells and heart tissues were assessed by using a GSH Detection Assay Kit (Cat#A006-2-1, Nanjing Jiancheng Bioengineering Institute, China). The kit is based on an enzymatic method in the presence of GSH which reacts with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) to form a 2-nitro-5-thiobenzoic acid (TNB) chromophore. The reduction of the chromophore releases a strong yellow signal which can be quantified spectrophotometriclly by using a microplate reader (OD = 405 nm) (Zhang et al., 2009 (link); Caito and Aschner 2015 (link)). Cells and tissues were similarly prepared, as mentioned previously (MDA assay). A total of 100 μL sample supernatant, a GSH standard solution (20 umol/L), and distilled water (standard blank) were mixed with 100 μg reaction buffer which contained DTNB, according to the manufacturer’s protocol. The mixture was kept on RT for 5min; then, the total mixture was used for the measurement of GSH concentration at OD = 405 nm. The GSH content (micromole per gram protein) was calculated by the same formula, as mentioned previously.