Pb180120

The human EC cell line (Ishikawa, CL-0283) was obtained from Procell Life Science &Technology (Wuhan, China). The cells were cultured in 90% phenol red-free DMEM (21063029, Gibco, Waltham, MA, USA), 10% charcoal dextran-treated FBS, and 1% P/S (PB180120) at 37 °C with 5% CO₂. Cells were treated with Diane-35 (Bayer Schering Pharma AG) and metformin (Sigma-Aldrich, St. Louis, MO, USA, CAS:1115-70-4). The Diane-35 tablets were milled into a powder using a crucible, then dissolved in DMSO, centrifuged in an ultracentrifuge (5000× g, 15 min), and the supernatant was taken and prepared into 1/2/5/10-mM solutions for cell treatment. metformin was prepared to the same concentrations using DMSO.
Ovarian granulosa cells were isolated from the ovaries of model mice. A PCOS and EC mouse model was constructed by the intraperitoneal injection of dehydroepiandrosterone (DHEA) and tumor cell transplantation (for further details, refer to the in vivo experiments section). Preantral follicles and small antral follicles were then punctured with a sterile puncture needle under a microscope (avoiding puncturing large sinus follicles). Ovarian granulosa cells were maintained in 90% DMEM (21063029, Gibco), 10% treated FBS, and 1% P/S (PB180120) at 37 °C with 5% CO₂.

Immune cells in the spleen were seeded into the 96-well plates (5 × 10⁵ cells per well) and incubated with PRP solution (4 mg/mL) in DMEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA, C11995500BT) containing 1% penicillin–streptomycin (Procell, PB180120) and 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 36 h of stimulation, the IFN-γ and IL-4 produced by immune cells were tested with the kits (Mouse IFN-γ precoated ELISpot kit and the Mouse IL-4 precoated ELISpot kit) (Dakewe Group, Shenzhen, China).