Isolation of Ovarian Granulosa Cells

As previously described (17 (link)), ovarian GCs were collected from follicular fluid on the oocyte retrieval day, the supernatant of follicular fluid was discarded after centrifuging at 2,000 rcf for 10 min at 4°C, 1 mg of hyaluronidase (Solarbio, Beijing, China) was added per 1 ml of Hanks balanced salt solution (Solarbio, China), and 1 ml of the solution was added to the sample tube. We then incubated the solution at 37°C for 30 min and added it to the upper liquid of 4 ml of human peripheral blood lymphocyte separation solution (TBDscience, Tianjin, China) and avoided mixing with the underlying liquid. We centrifuged the solution at 1,600 rcf for 10 min, collected the interphase, and went through preseparation filters (70 µm) (MACS, Frankfurt am Main, Germany) to eliminate cell clumps that may be inserted into the column. The mixture was centrifuged at 6,000 rcf for 5 min and the supernatant was discarded. We then transferred cells to a new microfuge tube by PBS resuspension and were then centrifuged at 6,000 rcf for 3 min. Fresh GCs were stored at −80°C after discarding the supernatant.