Total sod assay kit with wst 8

The SOD activity was determined using the Total SOD Assay Kit with WST-8 (Beyotime Bio., China), which is based on the ability of SOD to inhibit the oxidation of oxymine by O₂⁻ produced from the xanthine–xanthineoxidase system. Following the manufacturer's protocol, the SOD activity was measured at a wavelength of 550 nm. MDA, a product of lipid peroxidation, was also analyzed by an assay kit (Beyotime Bio., China), following the manufacturer's protocol. MDA levels were measured at 532 nm for the formation of stable chromophoric with thiobarbituric acid (TBA).

The plasma was separated from the blood samples by centrifugation at 10,000× g for 10 min. Tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) were quantified in plasma samples using a specific enzyme-linked immunosorbent assay (ELISA) with a mouse anti-mouse monoclonal antibody. The procedure complied with the manufacturer’s instructions (Fanyin, Shanghai, China).
The lung tissue was homogenized in PBS and centrifuged at 3500× g for 10 min, the supernatants were collected and dissolved in extraction buffer for the analysis of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) content. The total protein in the sample was determined using the BCA Protein Assay Kit (Solarbio, Beijing, China). To examine the level of lipid peroxidation in the lung tissue, the MDA concentration was assessed by the lipid peroxidation MDA Assay Kit (Beyotime, S0131, Shanghai, China). For antioxidative enzyme activities in the lung tissue, SOD and CAT levels were detected with a total SOD Assay Kit with WST-8 (Beyotime, S0101) and a CAT assay kit (Jiancheng Bioengineering Institute, A007-1-1, Nanjing, China).

As described previously, the cells were seeded in 6-well plates, treated with H ${}_2$ O ${}_2$ (500 $\mathsf{\mu }$ M) and GSH (1.6 mM). After the treatment, the lipid peroxidation MDA Assay Kit (S0131M; Beyotime Institute of Biotechnology) and total SOD Assay Kit with WST-8 (S0101M; Beyotime Institute of Biotechnology) were used to determined MDA levels and SOD activity, respectively, in cell homogenates according to the manufacturer’s instructions