S0056

The ovary was precisely weighed 0.2 g and homogenized in 2 mL of ice-cold PBS. After being centrifuged at 12,000 g for 10 min at 4°C, the supernatants were collected to measure the oxidative status. The protein content of the supernatants was measured with a BCA Protein Assay Kit (P0010, Beyotime Biotechnology, Shanghai, China). We assessed catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content in the ovary using commercial reagent kits (S0051, S0056, S0121 and S0131, Beyotime Biotechnology, Shanghai, China). All experimental procedures were performed according to the manufacturer's instructions. All results were normalized to protein concentration in each sample.

The kits used for the assessment of ROS (S0033), MDA (S0131), LDH (C0017), SOD (S0101), and GPx (S0056) in MC3T3-E1 cells were obtained from Beyotime Institute of Biotechnology. MC3T3-E1 cells were added into the corresponding wells, and the wells were sealed up by adhesive tape and maintained at 37°C for 90 min. And then 100 µl biotinylated antibody fluids were added into each well except for the blank wells. Afterwards, wells were sealed by adhesive tape and maintained at 37°C for 60 min. Plates were washed by PBS and 100 µl enzyme solutions were added into each well. After that, wells were sealed up with adhesive tape and maintained at 37°C for 30 min. Chromogenic substrate was added into the wells except for the blank wells. Plates were maintained for 10–15 min in the dark at 37°C. Afterwards, stop solution was added into each well, and mixed in 10 min immediately. Finally, the OD450 value was detected by microplate reader (Bio-Rad Laboratories, Inc.).