Gsh assay kit

GSH level was used to indicate the intracellular oxidative stress. The content of GSH was measured using a GSH Assay Kit (Keygen BioTECH, Nanjing, China). The GSH content was determined by reacting with 5, 5′-dithiobis-2-itrobenzoic acid (DTNB) to form a yellow-colored product, which can be measured by colorimetry. Briefly, SK-NSH cells were seeded in six-well plates (1×10⁶ cells/well) and treated with the designated experimental conditions. The cells were collected by 0.25% trypsin (w/v)/0.02% EDTA (w/v) and lysed in lysis buffer [0.05 mM EDTA, 1% Triton-X 100 (v/v), pH = 8.0] for 1 h at 4°C, and then the cell suspension was centrifuged at 12,000 rpm for 2 min. Subsequently, 1.9 ml freshly prepared disodium hydrogen phosphate buffer and 0.5 ml of 0.004% DTNB (w/v) was added into the supernatant. The optical density of the yellow-colored complex formed by the reaction of GSH and DTNB was measured by a spectrometer at a wavelength of 420 nm. The GSH content was calculated from a standard curve of GSH slope, and normalized to the protein concentration, which was determined by Coomassie Brilliant Blue method.