Sod activity assay kit

Ionizing radiation kills cells by increasing mitochondrial ROS. Cells contain antioxidants enzymes to prevent or repair the damage caused by ROS. The SOD activity assay was used to measure the antioxidant enzyme activity of SOD [56 (link)], using an SOD activity assay kit (Trevigen, Gaithersburg, MD, USA). Superoxide radical ions generate from the conversion of xanthine to uric acid, and H₂O₂ by xanthine oxidase converts WST-1 to WST-1 formazan. WST-1 formazan absorbs light at 450 nm. SOD reduces superoxide ion concentrations, thereby lowering the rate of WST-1 formazan formation. The extent of reduction in the WST-1 formazan represents of SOD activity level. The protein extract was used with the SOD activity assay kit. The pellets were isolated at 24 h after irradiation, and the reactions were initiated by adding xanthine solution at 25 µL/well in a 96-well plate. The plate was immediately transferred to a plate reader, and absorbance was measured at 450 nm every minute for 10 min at room temperature. The total SOD activity was calculated using the following equation: $$\% \mathrm{Inhibition}=\frac{\left(\mathrm{slope} \mathrm{of} 1\mathrm{X} \mathrm{SOD} \mathrm{buffer} \mathrm{control} - \mathrm{slope} \mathrm{of} \mathrm{sample}\right) }{\mathrm{slope} \mathrm{of} 1\mathrm{X} \mathrm{SOD} \mathrm{buffer} \mathrm{control}}\times 100$$

The PC12 cell-line was a gift from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT), and nerve growth factor (NGF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary anti-SOD2 and anti-cleaved caspase-3 antibodies were purchased from Santa Cruz (USA). MitoTracker and MitoSOX staining kits were purchased from Invitrogen Molecular Probes (San Diego, CA). The 4′,6-diamidino-2-phenylindole (DAPI) staining solution and ROS reagent kit were purchased from Beyotime Technology (Nantong, China). The SOD activity assay kit was purchased from Trevigen (Gaithersburg, USA). The LDH, GSH, and CAT reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).