Double antibiotics

PC-9, PC-9R, and MRC-5 cell lines were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum and 1% double antibiotics (Solarbio, Beijing, China) and then maintained in standard conditions (i.e., 5% CO₂ and 95% atmosphere, 37 °C). The medium was changed every three days and cells were passaged with 0.25% trypsin for digestion when the cell density reached 80% to 90% confluence.

The granulosa cells were isolated and cultured by Gilbert's established method (Gilbert et al., 1977 (link)). Briefly, the hierarchical follicles (6∼10mm in diameter) were obtained from White Leghorn hens, and then they were placed in phosphate-buffered saline (PBS) containing 3% double antibiotics (Solarbio, Beijing, China) to remove the residual connective tissue and attached blood filaments. The outer layer of the follicle membrane was peeled off with pointed tweezers. All granulosa cells were assembled in 1.5 mL centrifuge tubes, ground into a homogeneous paste by small tissue scissors, mixed in 15 mL centrifuge tubes, and digested in a 37°C environment with 5 mL 0.25% trypsin (Solarbio) for 8 min. After the digestion process, single cells were obtained by filtration through 200 μm sieve. After centrifugation twice, the suspended cells in Medium 199 (Gibco, Waltham, MA) complete culture medium containing 5% fetal bovine serum (Gibco) and 1% double antibiotics were spread in a 6-well plate. The number of cells was detected using trypan blue. Cells were cultured at 37.5°C in an atmosphere of water-saturated 5% CO_2.