Dmem f12

Peripheral lung tissue without the trachea and bronchi was mechanically dissociated with scalpels and digested by a solution of 2 mg/ml collagenase A, 2 mg/ml trypsin, and 50 μg/ml gentamicin (Sigma) in DMEM/F-12 (Gibco) for 45 min at 37°C with shaking. The digestion was stopped by the addition of 10% (v/v) fetal bovine serum (Gibco). The cell suspension was filtrated by a 100 μm cell strainer (Falcon) to eliminate tissue debris and cell clumps and was then collected by centrifugation at 4°C for 10 min. After being washed with DMEM/F-12 and undergoing centrifugation, the cell pellet was treated with red blood cell lysis buffer (Beyotime) 3 times for 2 min each. The cells were centrifuged again and resuspended in 20 U/ml DNase I (Sigma). After a final wash with DMEM/F-12, the cell suspension was filtrated through a 40 μm cell strainer (Falcon) and then seeded into nonadherent 25 cm² flasks pretreated with poly-HEMA (Sigma) in lungosphere medium [1×B-27, 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), 4 μg/ml heparin (STEMCELL), 20 ng/ml murine epidermal growth factor (mEGF; Peprotech), 10 ng/ml murine fibroblast growth factor 2 (mFGF2; Peprotech), and 10 μM Y-27632 (STEMCELL) in DMEM/F-12] (1.0×10⁶ cells/ml). The flasks were incubated in a humidified atmosphere at 37°C and 5% CO₂, and half the volume of medium was changed every 2 to 3 days.

Primary SCs were isolated according to previously described procedures [18 (link)–21 (link)]. In brief, trypsin (HyClone, Rockford, IL, USA) and collagenase IV (Sigma-Aldrich, St. Louis, MO, USA) were used to digest the scissored testes tissue blocks for 30–60 min at 37°C. After stopping the digestion by addition of 10% FBS (Gibco, USA) containing Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12, 1 : 1) medium (pH 7.2) (HyClone, Thermo Fisher Scientific, Rockford, IL, USA), the mixture was sieved through sterilized 100 meshes. Newly separated cells were cultured for 48 hours in 10 cm diameter dishes (Corning, New York, USA) with DMEM/F12, 10% FBS, and 1x penicillin-streptomycin (Beyotime Institute of Biotechnology, Haimen, China) in 5% CO₂ at 35°C. Then, cells were treated with 20 mM Tris-HCl (pH 7.4) for 2.5 min.