Sod assay kit

Frozen muscle samples were homogenized in cold saline and centrifuged at 12,000 g for 5 min. The supernatant was analyzed for total superoxide dismutase (SOD) activity by xanthine oxidase method with SOD Assay Kit (KeyGEN BioTECH, KGT001100) according to manufacturer’s protocol. Glutathione redox state was measured by total and oxidized glutathione (GSH and GSSG), respectively, using a commercially available kit (Solarbio BC1170 and BC1185).
Mitochondria of gastrocnemius were isolated with the Mitochondria Isolation Kit for Tissue and cell (invent, MP-007) according to the manufacturer’s instruction. Mitochondria content was tested by BCA assay. Mitochondrial membrane potential was determined by investigating fluorescence of 5,5,6,6-tetrachlore-1,1,3,3-tetraethylbenzimldazolylcarbocyanine iodide (JC-1) (Solarbio J8030, China) using a microplate fluorometer at an excitation/emission wavelength of 485/590 nm.

Cells were harvested and centrifuged at 2000 g, 4°C for 10 min, then the supernatant was removed. Washed by pre-cooled PBS, the same centrifugation was repeated. Cells were disrupted using ultrasound, centrifuged at 10,000 g, 4°C for 15 min, the supernatant were measured using the SOD assay kit (KeyGen, Shanghai, China) in accordance with the protocols. The OD value was determined at 550 nm.

Rats (n = 6 for each group) were sacrificed immediately postanesthesia (at PND 7), and the hippocampi were removed quickly. Intracellular ROS were detected using a ROS assay kit (Genmed Scientifics Inc., Shanghai, China) containing an oxidation-sensitive fluorescent probe (DCFH-DA) with a spectrofluorometer (excitation 490 nm, emission 520 nm). Malondialdehyde (MDA) is an end-product of ROS-induced peroxidation. Superoxide Dismutase (SOD) is an important enzyme that participates in the removal of ROS from the cellular environment. The extent of lipid peroxidation was estimated by MDA levels, which were measured by using the spectrophotometric diagnostic kits (Jiancheng Biological Technology Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. The SOD activity was determined using a SOD assay kit (Jiangsu KeyGEN BioTECH Co., Ltd., Nanjing, China) according to manufacturer’s instructions. Enzyme activity was converted to units per milligram of protein. One unit of SOD activity was defined as the amount that reduced the absorbance at 550 nm by 50%.

For cell cultures, DMEM, FBS, penicillin, streptomycin, and trypsin-EDTA were purchased from GE Life (HyClone, USA). DMSO, MTT, MTS, and Hoechst 33258 were purchased from Sigma (Santa Clara, USA). An annexin V-FITC/PI apoptosis detection Kit, reactive oxygen species (ROS) assay kit, malondialdehyde (MDA) assay kit, glutathione peroxidase (GSH-Px) assay kit and superoxide dismutase (SOD) assay kit were purchased from KeyGen Biotech (Beijing, CN).The primary antibodies against β-actin, Bax, Bcl-2, Caspase-9, Caspase-3, Cyt C, and Vadc 1 were purchased from Cell Signaling (Massachusetts, USA). Horseradish peroxidase-conjugated goat anti-rabbit antibody was also purchased from Cell Signaling and was used as the secondary antibody. All the other fine chemicals and solvents used for this study were of analytical grade.