Sod kit

According to the instruction on the SOD kit (Cayman Chemical, USA) provided by the manufacturer, the collected supernatant (10 μL) was mixed with the tetrazolium salt solution (200 μL) to dilute the SOD activity of the supernatants. The reaction was initiated by adding 20 μL of xanthine oxidase and incubated for 20 min in a shaker. The absorbance was then read using the ELISA reader at 440 nm. In this assay bovine erythrocyte SOD (Cu/Zn) was used as the standard. According to this assay procedure, xanthine oxidase and hypoxanthine detected superoxide radicals. In brief, the kit could measure the amount of enzyme that caused 50% dismutation of the superoxide radical.

Catalase (EC 1.11.1.6) and SOD (EC 1.15.1.1) activity were assayed from the same lysate sample and determined as per manufacturer's instructions (catalase kit and SOD kit, Cayman Chemical Company, Ann Arbor, MI). Absorbance values at 540 nm (catalase assay) and 450 nm (SOD assay) were determined via Model 680 Microplate Reader (Bio-Rad). For each time and concentration combination SOD n = 10–12 and catalase n = 5-6.

The assessment of SOD activity was conducted by SOD kit (Cayman Chemical, Michigan USA). The HuH-7 cells were seeded in 25 ml flask of 1 × 10⁶ cells/well for 24 h. Treatments were done as above h. Cells were washed, and 1 ml cold SOD lysis buffer (pH 7.2) was added. Cells were collected and sonicated for 2 min, then centrifuged at 12000 rpm at 4°C for 8 min. Then, 500 μl of supernatant was transferred into new Eppendorf tubes, and 200 μl of radical detector was added, followed by adding 10 μl of each sample (cell lysates), and then, 20 μl of diluted xanthine oxidase was added. The cells were incubated at RT for 10 min. OD was measured at 460 nm (Synergy-H1; BioTek).