Gsh gssg assay kit

HepG2 cells were seeded in 6-well plates at a density of 1 × 10⁵ cells per mL. The cells were treated with different SSeCAHK concentrations (0.13 mg mL⁻¹, 0.25 mg mL⁻¹, and 0.50 mg mL⁻¹) or FBS-free DMEM (control group) for 24 h, after which they were washed twice with PBS, and treated with 600 μM H₂O₂ for 2 h to induce oxidative stress. Next, the cells were washed three times with PBS and lysed with RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) at 4 °C for 10 min. The treated cells were collected and centrifuged at 4000 rpm for 10 min to obtain the supernatants, the MDA content of which was determined using an MDA assay kit (Nanjing Jiancheng Bioengineering Institute, China). The oxidized GSH (GSSG) and GSH content in the supernatants were determined using a GSH/GSSG assay kit (Nanjing Jiancheng Bioengineering Institute, China).

The GSH/GSSG assay was conducted using the GSH/GSSG assay kit (Jiancheng, Nanjing, China, A061-1-1) following the manufacturer's instructions. This method is based on the reaction between GSH and 5,5′-di-thiobis (DTNB), which leads to the cyclic production of TNB. The constant production of TNB is then measured using spectrophotometry at a wavelength of 405 nm. To perform the assay, a total of twenty-five cochlear explants were collected and homogenized using an ultrasonic homogenizer in 120 μL of the provided homogenization reagent on ice. The concentrations of total glutathione (tGSH) and oxidized glutathione (GSSG) were determined using a BioTek Synergy H1 reader (Agilent, Santa Clara, CA, U.S.). The levels of reduced glutathione (GSH) were calculated as follows: GSH = tGSH - (GSSG * 2). The concentration of GSSG is presented as nanomoles per 10 mg of tissue.