Intracellular ROS levels in H9c2 cells were quantified using a Reactive Oxygen Species Assay kit (Beyotime Institute of Biotechnology). After washing with PBS, the cells were suspended in 2′,7′-dichlorofluorescin diacetate (DCFH-DA) solution (10 µM) at 107/ml and incubated at 37°C for 20 min. The fluorescence intensity of DCFH-DA in the cells was detected by a fluorospectrophotometer (F-4000; Hitachi, Ltd., Tokyo, Japan).
The azino-diethyl-benzthiazoline sulfate (ABTS) method was adopted to examine the TAOC of H9c2 cells. Incubation with ABTS with H2O2 and a peroxidase (metmyoglobin) (provided by ABTS detection kit from Beyotime Institute of Biotechnology) results in the production of a blue-green radical cation ABTS+. Anti-oxidants contained in the H9c2 cells suppress this color production proportionally to the cells' TAOC. The system was standardized using Trolox (provided by ABTS detection kit), a water-soluble vitamin E analogue. The results were expressed as µmol Trolox equivalent/protein concentration of the H9c2 cells.
The azino-diethyl-benzthiazoline sulfate (ABTS) method was adopted to examine the TAOC of H9c2 cells. Incubation with ABTS with H2O2 and a peroxidase (metmyoglobin) (provided by ABTS detection kit from Beyotime Institute of Biotechnology) results in the production of a blue-green radical cation ABTS+. Anti-oxidants contained in the H9c2 cells suppress this color production proportionally to the cells' TAOC. The system was standardized using Trolox (provided by ABTS detection kit), a water-soluble vitamin E analogue. The results were expressed as µmol Trolox equivalent/protein concentration of the H9c2 cells.