Sod assay kit with wst 8

Total superoxide dismutase (SOD) Assay Kit with WST-8 (S0101) was purchased from Beyotime Co. (Shanghai, China) and the assay was performed according to the manufacturer’s instructions. The activity of NADPH oxidase was detected by commercial kits (Shanghai, China).

Chinese pecans were purchased from Lin’an Tongda Food Co., Ltd. (Hangzhou, China). Compound protease (120 U/mg) and glutathione (GSH) were obtained from Chinese Shanghaiyuanye Bio-Technology Co., Ltd. (Shanghai, China). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was bought from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Sephadex G-25 was obtained from Beijing Solarbio Biotechnology Co., Ltd. (Beijing, China). H₂O₂ was obtained from Sigma-Aldrich (Shanghai, China) Trading Co., Ltd. (Shanghai, China). Caco-2 cells were acquired from the Chinese Academy of Sciences (Kunming, China). Cell Counting Kit-8 (CCK-8), ROS assay kit, Catalase assay kit, and SOD assay kit with WST-8 were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China). All the reagents were analytical grade.

Protein extracts from the treated cells and tumor tissues were subjected to measure MnSOD activity using a SOD Assay Kit with WST-8 (Beyotime Biotechnology) according to the manufacture’s instruction. Briefly, the extracts were centrifuged at 12000 rpm at 4 °C for 10 min to remove cell debris and the supernatants were added to a WST-8/Enzyme working buffer containing a Cu/ZnSOD inhibitor at 37 °C for 30 min. The OD values were measured at 450 nm using a microplate reader. The activity of MnSOD was calculated according to the formula provided by the manufacture.

SOD Assay Kit with WST-8 (Beyotime) was used to assess SOD activity in CPB2 toxin-infected IPEC-J2 cells. The cell culture was aspirated and washed once with pre-chilled PBS at 4 °C. A total of 200 μL of the SOD sample preparation was added to each well to fully lyse the cells. After centrifugation at 12,000× g for 5 min at 4 °C, the supernatant was transferred to a 96-well plate (20 μL/ well). Subsequently, 160 μL of WST-8/enzyme working solution and 20 μL of reaction starter working solution were added to each well, gently mixed and incubated for 30 min at 37 °C. Finally, the absorbance was measured at 450 nm on a multifunctional enzyme marker (Molecular Devices).