Isolation of Ovarian Granulosa Cells
Ovarian GCs were collected from follicular fluid which is aspired on the oocyte retrieval day. Follicular fluid was centrifuged at 2000rcf for 10 minutes at 4°C and the supernatant was discarded. Added 1mg of Hyaluronidase (Solarbio, China) per 1ml of Hanks Balanced Salt Mixture (Solarbio, China) and 1ml of the mixture was added to the sample tube. Then incubated the mixture at 37℃ for 20 minutes. Added the mixture to the upper liquid of 4 ml of Human Peripheral Blood Lymphocyte Separation Solution (tbdscience, China) and avoided to mix with the underlying liquid. The mixture was centrifuged at 1700rcf for 10 minutes. Collected the middle liquid containing GCs and went through a Pre-Separation Filters (70 µm) (MACS, Germany) to eliminate cell clumps that may be inserted into the column. The mixture was centrifuged at 6000rcf for 5 minutes and discarded the supernatant. The cells were transferred to a new microfuge tube by PBS resuspending. The new microfuge tube with GCs suspension was centrifuged at 6000rcf for 3 minutes. Fresh GCs were stored at −80°C after discarding the supernatant.