Lipid peroxidation malondialdehyde mda assay kit

The myocardium (H9C2 cells) total ROS, oxidative stress, and antioxidant levels toward hypoxia, HIF-1α inhibition, and under different treatments were measured using commercially available assay kits by following the manufacturer’s protocol. Intracellular total ROS levels were measured using a 2′-7′-dichlorofluorescein diacetate-based total ROS detection kit (#ENZ-51011, Enzo Life Sciences). Oxidative stress levels were assayed using Protein Carbonyl assay using the bioluminescent assay kit (MAK135; Sigma-Aldrich, United States) by following the manufacturer’s protocol and as previously described by Osuru et al. H9C2 cells Lipid peroxidation level was determined using the thiobarbituric acid (TBA) method with Lipid Peroxidation [malondialdehyde (MDA)] assay kit (MAK085; Sigma-Aldrich United States). H9C2 cell’s antioxidant levels were measured by quantifying the total SOD (Superoxide dismutase) levels using the SOD colorimetric activity kit (#EIASODC; Life technologies corporation-Invitrogen, United States) and by estimating the catalase (CAT) protein levels using western blotting method (for antibody concentration and methodology please see the western blotting section).

Aiming to determine the radical scavenging capacity, W-SE and MWT-SE from samples were assessed. DPPH radical scavenging activity was measured by using the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) as reported above. ABTS radical scavenging activity was estimated by the Antioxidant Assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. The principle of ABTS assay is the formation of a ferryl myoglobin radical from metmyoglobin and hydrogen peroxide, which oxidizes the ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) to produce a radical cation, ABTS.⁺, a soluble chromogen that is green in color and can be determined spectrophotometrically at 405 nm. Trolox, a water-soluble vitamin E analog, was used as a control antioxidant. Lipid peroxidation was calculated using the Lipid Peroxidation Malondialdehyde (MDA) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. In this kit, lipid peroxidation was determined by the reaction of MDA with thiobarbituric acid (TBA) to form a colorimetric (532 nm) product, in proportion to the MDA present.