Superoxide dismutase colorimetric activity kit

Total SOD activity was measured using the Superoxide Dismutase Colorimetric Activity Kit (Invitrogen), according to the manufacturer’s protocol. Briefly, H9c2 cells from the different treatments were sonicated in cold PBS and the supernatant was used for the assay. The reaction was initiated by adding xanthine oxidase. SOD activity absorbance was measured at 450 nm on an Infinite M200PRO microplate reader (Tecan Group Ltd., Männedorf, Switzerland) and expressed as U/mg of total proteins. One unit of SOD is defined as the amount of enzyme causing half the maximum inhibition of 1.5 mmol L⁻¹ blue tetrazolium reduction in the presence of riboflavin at pH 7.8 and 25 °C. Total protein content was measured spectrophotometrically using micro-BCA™ Protein Assay Kit (Pierce, IL, USA).

Superoxide dismutase (SOD) activity was measured by the superoxide dismutase colorimetric activity kit (Invitrogen Co., Waltham, MA, USA). RAW 264.7 cells were seeded in a 6-well plate at a concentration of 2 × 10⁵ cells/well. After incubation at 37 °C for 4 h, AHL and AHR extracts were treated in the RAW 264.7 cells at different concentrations (100, 250, and 500 μg/mL) and were incubated at the same condition for 48 h. The supernatant of media was collected by centrifugation at 1500 rpm for 10 min at 4 °C. In brief, each 10 μL of supernatant was added to 50 μL of the substrate solution with 25 μL of a xanthine oxidase solution in a new plate and the plate was kept at RT for 20 min. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices). The SOD activity was calculated by the standard curve.