Superoxide dismutase activity assay kit

SOD activity was measured using a Superoxide Dismutase Activity Assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. The volume of the solution used in the experiment was halved. Then, 10 μL of the cytosolic fraction was used without dilution. The assay utilized a water-soluble tetrazolium salt (WST-1) that produced a water-soluble formazan dye upon reduction with a superoxide anion. SOD catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, resulting in a decrease in the levels of WST-1, which has an absorbance at 450 nm. The inhibition of SOD activity was recorded using a SpectraMax iD3 (Molecular Devices, San Jose, CA, USA). The inhibition rate of the sample was normalized by dividing the absorbance unit by the weight of the sample.

The total concentrations of GSH and oxidized glutathione (GSSG) in heart tissue or cultured cells were determined with the Colorimetric GSH Assay Kit (ab239709, Abcam, Cambridge, UK) following the manufacturer's protocol. Total SOD activity of heart tissue homogenates or cell lysates was detected with Superoxide Dismutase Activity Assay Kit (ab65354, Abcam) according to the manufacturer's instructions.

To evaluate the anti-oxidative effect of Hitrechol®, catalase (CAT) activities, and superoxide dismutase (SOD) and reduced glutathione (GSH) levels were detected in collect serum and prepared liver homogenate from Groups 1 to 5 using Catalase Activity Assay Kit (ab83464), Superoxide Dismutase Activity Assay kit (ab65354) and GSH/GSSG Ratio Detection Assay kit (ab138881) according to the instructions of the manufacturer (Abcam, USA). Furthermore, to evaluate the anti-inflammatory effect of Hitrechol®, the concentration of TNF-alpha in serum were detected using ELISA kit (Abcam, USA).

LV tissues were homogenized in ice-cold 0.1 M Tris/HCl, pH 7.4 containing 0.5% Triton X-100, 5 mM mercaptoethanol and 0.1 mg/ml PMSF using a TissueLyser II (Qiagen). The crude tissue homogenates were then centrifuged (14,000×g, 5 min, 4°C) and the supernatants collected: these contained total SOD activity from combined cytosolic and mitochondrial sources. Following incubation with 0.4 vol of a solution containing ethanol and chloroform (25:15 vol/vol) to inactivate the mitochondrial SOD2 [59 (link)], mixtures were centrifuged at 5000 × g for 15 min and aliquots of the supernatant used to measure protein concentration by BCA assay, and Cu/Zn-SOD activity using the Superoxide Dismutase Activity Assay kit (Abcam). The latter works by measuring the inhibition of the reduction of a water-soluble tetrazolium salt (WST-1) which produces a water-soluble formazan dye upon reduction by O₂^●- radicals, which are generated by xanthine oxidase and inhibited by SOD. One unit of SOD activity is the amount necessary to inhibit the xanthine oxidation by 50%. The IC₅₀ (50% inhibition activity of SOD) was detected by a colorimetric method at 450 nm using an absorbance microplate reader (SpectraMAX 340, Molecular Devices) and the results expressed per mg of total protein.

According to manufacturer protocol, the levels of SOD/ROS in PC-12 cells or brain tissues of rats were measured using Superoxide Dismutase Activity Assay Kit or Reactive Oxygen Species Assay Kit (Abcam, Cambridge, MA, USA), respectively. SOS activity (OD450) and ROS fluorescence (OD520) were measured using a Synergy HT microplate reader (Biotek, Winooski, VT, USA).