S0119

Reactive oxygen species (ROS) were detected by a dichlorodihydrofluorescein diacetate (DCFH-DA) probe with a BioTek Gen 5 instrument (Ex 488 nm, Em 525 nm) and a fluorescence microscope (Leica DMI4000B, Germany). Myeloperoxidase (MPO) activity was measured using a commercial kit (A044-1-1, Jiancheng Biotech) according to the manufacturer’s instructions. The total antioxidant capacity in lung tissues/cell homogenate was measured using a commercial kit (S0119, Beyotime). Superoxide dismutase (SOD) activity was measured using a kit (S0109, Beyotime) based on the nitroblue tetrazolium reduction reaction.

The assay for total oxidative capacity in serum was performed according to the manufacturer’s instruction (Beyotime Biotechnology, S0119, Shanghai, China). Briefly, 20 µL of peroxidase solution was added to each well of the 96-well plate, and 10 µL of PBS or serum was then added to the control or the tested wells and mixed. Secondly, we added 170 µL of ABTS solution to each well, which were then mixed gently, incubated for 6 min at room temperature and the OD value was measured at 414 nM.

An appropriate amount of ovarian tissue sample was added to prepare the solution. Then, a hand-held electric homogenizer was used to homogenize the samples on ice, and the supernatant was collected as the sample to be tested. The protein concentration was determined with a BCA protein concentration kit for quality control for subsequent detection. The working solution was prepared according to the instructions of the kit, the sample was diluted, the 96-well plate was gently shaken to fully mix the solution, the plate was incubated in an incubator at 37 °C, and the absorbance of the sample was detected at 450 nm. The enzyme activity was calculated according to the instructions of the kit {CAT (Beyotime, S0082, Shanghai, China), SOD (Beyotime, S0101S, Shanghai, China), GSH-Px (Beyotime, S0056, Shanghai, China), total antioxidant capacity (T-Ait isOC, Beyotime, S0119, Shanghai, China) and MDA (Beyotime, S0131S, Shanghai, China)}.