Reactive oxygen species (ROS) assay

Intracellular ROS levels were evaluated using a cellular ROS/superoxide-detection assay kit (Beyotime, Haimen, China), according to the manufacturer’s instructions. In brief, cells were seeded in 96-well plates at a density of 10⁵ cells/well and allowed to attach for 24 hours. After incubation with the aforementioned treatments, the culture supernatant was removed and the cells washed with 100 μL/well of assay buffer. The ROS-specific stain DCFH-DA was added to the cells and allowed to incubate in the dark for 60 minutes. After incubation, intracellular ROS levels were determined using a fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 520 nm).