Enzyme-linked immunosorbent assay (ELISA) for the measurement of inflammatory cytokines and markers of oxidative stress

The enzyme-linked immunosorbent assay (ELISA) was used to measure reactive oxygen species (ROS), including malondialdehyde (MDA), nitric oxide (NO), p-eNOS, and the inflammatory cytokines, tumor necrosis factor-α (TNF- α), interleukin-1β (IL-1β), IL-6, and monocyte chemoattractant protein-1 (MCP-1) in H9c2 cell supernatants. The measurements were performed according to the manufacturer’s instructions for the Duo-Set enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA). The optical density (OD) value at 450–650 nm was determined by using a microplate reader.

The levels of malondialdehyde (MDA) and nitric oxide (NO) produced by H9c2 cells were measured by the enzyme activity assay kits (Beyotime, Shanghai, China) according to the manufacturer instructions. Briefly, samples (0.1 mL) and mixed reagent (0.4 mL) were added and mixed evenly in a 6-well plate. The concentrations of reactive oxygen species (ROS) in the cell supernatants was analyzed by 2′-7′-dichlorofluorescein diacetate (Beyotime, Shanghai, China). The cells were stained with 20 μM of dichloro-dihydro-fluorescein diacetate (DCFH-DA) at 37°C for 40 min in the dark. The absorbance was measured by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). For lactate dehydrogenase (LDH), glutathione peroxidase (GSH-Px), and the superoxide dismutase (SOD) activity assay, H9c2 cells were disrupted and centrifuged to obtain the cell supernatant. The supernatant was mixed and incubated with the reaction buffer at 37°C for 30 min. The absorbance was measured using a microplate reader.