Sod assay kit wst

The tissues were lysed with a lysate buffer and then centrifuged at 12,000 g at 4°C for 5 min. Afterward, an aliquot of the supernatant was used for detection. Superoxide dismutase enzyme activities were measured using a commercial SOD Assay Kit-WST (Beyotime Biotechnology, CHN) in accordance with the manufacturer’s protocol. The values were recorded using a microplate reader (CLARIOstar, BMG LABTECH, GER). The concentration of total proteins in the cells was quantified by using the BCA Kit (Takara, JP).

After protein purification as mentioned above, 1–2.5 μg protein was used to measure superoxide dismutase (SOD) or catalase activities. Precisely, SOD activity was determined with a SOD assay Kit-WST (Beyotime, China). The Catalase Assay Kit (Beyotime, China) was used to detect catalase activity. Briefly, for SOD activity assay, the samples were treated by WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium, monosodium salt] and incubated at 37°C for 30 min. Afterward, the absorption maximum at 450 nm was measured by Thermo Multiskan EX Micro plate Photometer (Thermo Fisher Scientific, Waltham, MA, USA). SOD activity was calculated by standard curve according to the instruction of manufacturer in kit. For catalase assay, the samples were treated with 10 μl of 250 mM H₂O₂ and incubated 5 min at room temperature, and the remaining H₂O₂ (not decomposed by catalase) was coupled with a substrate to generate N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquinonemonoimine, which has an absorption maximum at 520 nm and was quantified spectrophotometrically. Catalase activity was then calculated by standard curve according to the instruction of manufacturer in kit. These experiments were repeated three times independently.