Sod assay kit

The SOD activity in the PC12 cells after OGD was measured using a SOD assay kit (Cayman Chemical, Ann Arbor, MI, USA). Tetrazolium salt, xanthine oxidase and cell lysate were added to a 96-well plate. The OD540 value of the formazan produced was measured by a fluorescent microplate reader (FLUOstar OPTIMA, BMG LABTECH, Ortenberg, Germany). The results of these experiments were expressed as the relative percentage of SOD activity of each sample compared with the control.

PBLEs effects on the activity of the endogenous antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and thioredoxin (Trx), were assessed using the appropriate assay kits.
SOD is a critical player in the antioxidant defense mechanism in cells that converts the superoxide anion to molecular oxygen and hydrogen peroxide. Superoxide radicals produced by xanthine oxidase and hypoxanthine are detected using the SOD assay kit (Cayman Chemical, catalog no. 706002, Ann Arbor, MI, USA), and SOD activity was expressed as U/mg protein.
CAT detoxifies the cell-toxic hydrogen peroxide. The activity of CAT was measured using a CAT assay kit (Cayman Chemical, catalog no. 707002, Ann Arbor, MI, USA), following manufacturer instructions, and was expressed as U/mg protein.
The GSH assay kit (Cayman Chemical, catalog no. 703002, Ann Arbor, MI, USA) allows for the measurement of total GSH. GSH produces the yellow 5-thio-2-nitrobenzoic acid (TNB) as a byproduct of the reaction with 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB). The rate of TNB synthesis is directly proportional to the GSH formation, which is quantified at 405–414 nm and represented as mol/mg protein.
For thioredoxin (Trx) activity, ELISA kit (MyBioSource, Inc., catalog No: MBS2019144, San Diego, CA, USA) was used, according to the manufacturer’s instructions.

The hepatic SOD activity was determined by using a commercialized chemical SOD assay kit (Cayman Chemical Co.). The kit utilizes a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. The collected 1 gm of liver tissues were homogenized in 5 ml of cold buffer (20 mM HEPES buffer, pH 7.2, containing 1 mM EDTA, 210 mM mannitol, and 70 mM sucrose) and centrifuged at 1,500 × g for 5 min at 4°C. The reaction was initiated by adding xanthine oxidase and incubating at room temperature for 20 min, and then, the absorbance of each sample was read at 450 nm.

A superoxide dismutase (SOD) assay kit (Cayman chemical) was used to measure MnSOD (SOD2) activity in mouse brain. Pre-weighed, perfused mouse brain pieces were homogenized in 5 mL cold 20 mM HEPES buffer, pH 7.2, supplemented with EGTA, mannitol and sucrose, with a Dounce homogenizer on ice. The homogenates were centrifuged at 1500×g 5 min at 4 °C. The resulting supernatants were centrifuged at 10,000×g 15 min at 4 °C to isolate the mitochondria (pellet). Two mM potassium cyanide, which inhibits Cu/Zn-SOD and extracellular SOD, was added to each sample to ensure assay specificity for MnSOD. The SOD standards were prepared by adding 200 μL of the radical detector and 10 μL of the provided standards, in duplicates in a 96-well plate. The same was repeated for the samples. The reaction was initiated by adding 20 μL of xanthine oxidase to all the wells. Background absorbance was assayed by adding 20 μL xanthine oxidase to sample buffer (optional). The plate was incubated on a shaker for 30 min at room temperature. The absorbance was measured at 450 nm. The linearized SOD standard curve was plotted and used to calculate MnSOD activity (U/mL) from averaged sample absorbance readings.