Bovine intramuscular preadipocytes isolation

Dabieshan yellow cattles (24–30 months old, male) were provided by Hubei Hegen Agricultural Technology Ltd and harvested at a local abattoir using standard procedures. Bovine intramuscular preadipocytes were isolated from longissimus dorsi muscle, the method was as follows. The longissimus dorsi muscle was washed five times with PBS containing 5% penicillin/streptomycin and transported to laboratory in PBS. The following procedures were conducted in a sterile field. Adipose tissues were separated from muscle bundles and finely chopped into 1-mm³ pieces with scissors in PBS and then incubated with 0.1% collagenase type I (Sigma) for 1 h at 37°C with mixing every 10 min. After enzymatic digestion, the released fat stromal cells were suspended in DMEM (Gibco) supplemented with 15% fetal bovine serum (FBS; Gibco), and the suspension was filtered through a 100 µm filter (Corning Incorporated). Then, the cells were collected by centrifugation at 650 g for 5 min. The cells were added to fresh DMEM supplemented with 15% FBS and 1% penicillin/streptomycin. The cells were then plated in nunclon flasks and cultured in an atmosphere of 5% CO₂ at 37°C. After 12 h, the non-adherent cells were removed. When cells achieved 80% to 90% confluence, they were passaged by trypsinisation.