Cu zn sod and mn sod assay kit with wst 8

Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) enzymatic activities were determined by using Cu/Zn-SOD and Mn-SOD Assay Kit with WST-8, Catalase Assay Kit, and Total Glutathione Peroxidase Assay Kit (Beyotime, Beijing, China) following the manufacturer’s instructions. Briefly, SOD activity determination was based on the inhibition of the superoxide radical-dependent cytochrome C reducing measured at a wavelength of 450 nm using Infinite M200 PRO Multimode Microplate Reader. CAT activity determination was based on the reducing absorbance at 520 nm due to the ability of scavenging H₂O₂, and the enzyme activity was converted by the speed of H₂O₂ consumption based on a standard curve obtained by the scalar units testing. GPx activity was determined according to that the speed of NADPH decrease was proportional to GPx activity measured at 340 nm using Infinite M200 PRO Multimode Microplate Reader. Enzyme activities were expressed as a percentage of control.

Mitochondria from mouse myocardium tissue and NRVMs were processed as group divided. According to the manufacturer’s protocols, SOD2 enzymatic activity was evaluated by a Cu/Zn-SOD and Mn-SOD Assay Kit with WST-8 (Beyotime, China), based on the capacity of SOD2 to competitively inhibit WST-8 by combining with superoxide radicals generated by xanthine oxidase. SOD1 inhibitors A and B were added to the sample to surpass the residual SOD1 activity, then samples were mixed with WST-8/enzyme working solution for 30 min at 37 °C. The absorbance of 450 nm was detected. When WST-8 formazan inhibition rate is 50%, SOD2 enzyme activity is defined as 1 unit. The protein concentration was analyzed by the BCA standard curve.

The SOD2 activity assay was performed using the Cu/Zn-SOD and Mn-SOD Assay Kit with WST-8 (Beyotime, China) according to the manufacturer’s instructions. After the cochlear tissue was cut and digested with trypsin, the cells were extracted by centrifugation. Cells were incubated for 1 h at 37°C using Cu/Zn-SOD inhibitor A and for 15 min at 37°C using Cu/Zn-SOD inhibitor B. Consequently, samples were added to a 96-well plate and mixed with the assay solution. Chemiluminescence was measured using an EnSpire enzyme marker (PerkinElmer). SOD2 viability units were calculated using a standard curve.