Sod activity

MnSOD activity was measured in PBMCs using commercially available kit (SOD activity Enzo Life Sciences, USA). The assay is based on the principle that MnSOD neutralizes superoxide anions produced by the xanthine/xanthine oxidase system and subsequently inhibits the reduction of WST-1 (water soluble tetrazolium salt) to WST-1 formazan. Briefly, PBMCs were harvested, washed with ice-cold 1x PBS, and lysed as described in kit protocol. Preincubation of the cell lysate with 2 mM potassium cyanide (KCN) was performed to inactivate both Cu/Zn-SOD and extracellular SOD.
It is important to note that the linear range of the assay was evaluated by generating a standard curve prior to measuring the MnSOD activity. Specifically, we created a serial dilution of the cell extracts, ranging from 0.5 μg/25 μL to 50 μg/25 μL, with 1x SOD buffer. Afterward, the percentage of inhibition of the formation rate of WST-1 formazan was compared with a standard curve generated with serial dilutions of the SOD standard. The SOD standard, WST-1 reagent, and 1x SOD buffer were supplied in kit form. Protein concentration was determined using Bradford method [20 (link)]. MnSOD activity values were expressed as units/mg of protein.

The SOD activity was measured using commercially available kit (SOD activity Enzo Life Sciences, Farmingdale, NY, USA). The principle of the method is based on the ability of SOD to neutralize superoxide ions created by the xanthine/xanthine oxidase system and subsequently inhibit the reduction of WST-1 (water soluble tetrazolium salt) to WST-1 formazan. Briefly, MC3T3 cells (5 × 10⁶ in six-well plate), obtained 6 h after oxidative stress induction, were washed with ice-cold 1× PBS, and lysed as described in kit protocol. The supernatant of each sample was collected, and the total SOD activity was assayed spectrophotometrically at 450 nm.