2.7. Detection of ROS and MDA

The ROS detection assay kit (CA1410, Sulaibio, Beijing, China) is designed for the detection of reactive oxygen species in live cells, and it is one of the most commonly used methods based on the change of fluorescence intensity of 2,7-dichlorofluorescin diacetate (DCFH-DA), which is easily oxidized into fluorescent dichlorofluorescein (DCF) by ROS. The cells were harvested by centrifugation (6000× g, 5 min) and resuspended in 10 μM DCFH-DA for 30 min at 37 °C in the dark. Subsequently, they were centrifuged and resuspended in 40 μL PBS buffer, followed by fluorescence microscopy using an excitation wavelength of 500 nm and an emission wavelength of 525 nm [15].

The MDA concentration was determined using the MDA assay detection kit (BC0025, Sulaibio, Beijing, China) based on thiobarbituric acid, and the procedures were carried out in accordance with the kit instructions. The reaction assays were incubated in a water bath at 95 °C for 40 min, after which the absorption of the MDA reaction product was measured at a wavelength of 532 nm [15].