Gsh detection kit

H9c2 whole cell lysates were collected according to the manufacturer's instructions using RIPA lysis buffer. The activities of SOD, MDA, and GSH were investigated using the corresponding kits. The MDA level was determined using the thiobarbituric acid method and MDA detection kit (A003-1-2; Jiancheng Bioengineering Institute). SOD and GSH activity were detected using the hydroxylamine method, and total SOD detection kit, and GSH detection kit (Jiancheng Bioengineering Institute).

For the measurement of GSH content, HepG2 cells (2.5×10⁵ cells/well) were seeded in 6-well plates and grown overnight. After exposure to triptolide (10, 20, 40, 80 nM) for 6 h, the treatment cell pellets were lysed by ultrasonication. Following centrifugation (4×10³ g for 10 min at 4°C), the supernatant (cell extract) was maintained on ice until assayed for the cellular GSH by a GSH Detection Kit (Nanjing Jiancheng Bioengineering institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions.

Unless otherwise mentioned, all chemicals used were purchased from Sigma–Aldrich. Dimethyl sulfoxide (DMSO) was used to create BPA stock solutions at different concentrations (20, 50, and 100 μg/mL). The final concentration of DMSO in the culture system was set at 0.1%, and the final BPA dosage used was determined on the basis of our preliminary experiment and a previous report [16 (link)]. Ultra-pure water was used to dissolve the NAC stock (200 mM). The final dose of NAC was 100 μM according to our preliminary experiment (see Additional file 1: Figure S1) and a previous report [41 (link)]. The ROS Assay Kit was purchased from Beyotime Institute of Biotechnology (Nantong, China), and the GSH detection kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The maturation medium was tissue culture medium-199 (TCM199; Gibco) supplemented with 10% (v/v) foetal bovine serum (Gibco), 24.2 mg/mL sodium pyruvate, 0.05 IU/mL follicle-stimulating hormone, 1 μg/mL 17β-oestradiol, 0.05 IU/mL luteinizing hormone and 10 ng/mL epidermal growth factor. Human tubal fluid (HTF; Merck Millipore) was used for sperm capacitation. KSOM medium (Merck Millipore) was used for embryo culture.

The detection of GSH in the liver tissue was performed using a GSH detection kit (Jiancheng, Nanjing, China). The tissue was carefully and accurately weighed according to the ratio of weight (g): volume (mL) = 1:9, and normal saline was added to make the tissue homogenate. The sample was centrifuged at 2500 rpm for 10 min; next, the supernatant was collected, mixed with the reagent in a ratio of 1:1, and centrifuged at 3500 rpm for 10 min. Finally, the supernatant was collected, and the corresponding reagents were added according to the manufacturer’s instructions, mixed well, and left to stand for 5 min. Each well’s absorbance was measured at 405 nm by the microplate reader.

A GSH detection kit (Nanjing Jiancheng) was used for cell GSH detection. After cell intervention, the medium was removed and the cells were washed once with PBS. PBS was added and the cells were scraped into a centrifuge tube. The cells were broken by sonication. The working solution was added, mixed, and allowed to stand for 5 minutes; after which, the absorbance of each well was measured at 405 nm wavelength using a microplate reader.

Activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase(GPx) of homogenized liver were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). GSH was also measured using a GSH Detection Kit (Nanjing Jiancheng Bioengineering institute, Nanjing, Jiangsu, China) according to the manufacturer's instructions.