Total sod detection kit

H9c2 whole cell lysates were collected according to the manufacturer's instructions using RIPA lysis buffer. The activities of SOD, MDA, and GSH were investigated using the corresponding kits. The MDA level was determined using the thiobarbituric acid method and MDA detection kit (A003-1-2; Jiancheng Bioengineering Institute). SOD and GSH activity were detected using the hydroxylamine method, and total SOD detection kit, and GSH detection kit (Jiancheng Bioengineering Institute).

The ROS production, malondialdehyde (MDA) level, SOD and catalase (CAT) activity, and Fe²⁺ in the cells under different treatments were measured using the corresponding kits. The cells were incubated with 2,7‐dichlorodihydrofluorescein diacetate (DCFH‐DA) probe in the dark for 30 min in line with the instructions of the ROS detection kit (Beyotime, Shanghai, China). Then the fluorescence intensity was observed under the fluorescent microplate. The laser wavelength was 485 nm, and the emission wavelength was 525 nm; ROS level (%) = fluorescence value of intervention group/control group × 100%. CAT activity was detected using visible spectrophotometry and CAT detection kit (A007‐1‐1; Jiancheng Bioengineering Institute, Nanjing, China). MDA level was determined using the thiobarbituric acid method and MDA detection kit (A003‐1‐2; Jiancheng Bioengineering Institute). SOD activity was detected using the hydroxylamine method and total SOD detection kit (A001‐1‐2; Jiancheng Bioengineering Institute). The absorbance value at 593 nm was measured to calculate the iron ion level in line with the instruction of Fe²⁺ iron ion kit (MAK025; Sigma‐Aldrich).

Formic acid (HPLC grade) and acetonitrile (HPLC grade) were purchased from Fisher (Waltham, Massachusetts, USA). Dulbecco's Modified Eagle Medium (DMEM) was acquired from HyClone Laboratories. Fetal bovine serum (FBS) was purchased from Sigma, so were 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), and 2,7-dichlorofluorescin diacetate (DCFH-DA). Annexin FITC/PI Apoptosis Detection Kit was procured from MultiSciences Biotech Co., Ltd. (Hangzhou, Zhejiang, China). Total SOD Detection Kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). M-MLV was obtained from Promega Corporation, and SYBR^R Premix Taq™ from TaKaRa. Protease/phosphatase inhibitor cocktail was purchased from Roche (USA). Antibodies specific for Bax, Bcl-2, Caspase-3, and p53 were procured from Santa Cruz. ERK, p-ERK, AKT, p-AKT, β-actin, and HRP-conjugated secondary antibody were purchased from Cell Signaling Technology.