Dme f 12 medium

Sheep ovaries obtained from a local slaughterhouse (Baoding, China) were placed in saline at about 37 °C and transported to the laboratory within 3 h. After washing the ovaries with saline, follicles from 3 to 6 mm in diameter were sliced, and cumulus–oocyte complexes (COCs) were obtained from the drained off follicular fluid. Then, the follicular fluid was collected into saline in a sterile environment, centrifuged, and cells were suspended in complete medium. Subsequently, granulosa cells were cultured in DME/F-12 medium (Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) at 38.5 °C in a humidified atmosphere of 95% air and 5% CO₂. As for COCs, only those with three or more layers of cumulus and uniform ooplasm were selected, and then they were cultured in TCM199 supplemented with 10% FBS, 10 mg/mL FSH, 10 mg/mL LH, 1 mg/mL E₂, 1 mmol/L L-glutamine, and 20 ng/mL EGF for 24 h at 38.5 °C and 5% CO₂ in humidified atmosphere.

The murine HCC cell line HCA-1 and the human HCC cell line JHH-7 were kindly provided by Dan Duda, Massachusetts General Hospital, Boston, MA. The HCA-1 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Corning). The JHH-7 cells were cultured in DME/F12 medium (Corning), and CCRF-CEM (T cell acute lymphoblastic leukemia; ATCC) cells were kept in RPMI-1640 medium (Gibco). The culture media were supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin and streptomycin (HyClone). The cells were maintained at 37 °C in an incubator (Thermo Fisher Scientific) with an atmosphere of 5% CO₂. HEK293T cells (ATCC) were kept in DMEM (Gibco).