Isolation of Muscle Cells from Beef and Dairy Breeds

To isolate muscle cells, samples of semitendinosus muscle were thawed immediately in a water bath and subsequently washed in PBS with Penicillinum crystallisatum. After PBS aspiration, the incubation medium (Dulbecco’s modified Eagle medium (DMEM) (Gibco, Life Technologies, Carlsbad, CA, USA), Pronase from Streptomyces griseus (Sigma-Aldrich, Saint Louis, MO, USA) at 0.5 mg/mL, 10% FBS, Penicillinum crystallisatum) was added. Samples were incubated for 1.5 h at 37 °C with constant mixing. Afterwards, the suspension with isolated muscle cells was sieved through a 70-µm nylon filter to separate tissue debris. The filtrate-containing cells were centrifuged three times and resuspended in proliferation medium (10%FBS/DMEM/1% penicillin–streptomycin and 0.5% amphotericin B (Gibco, Life Technologies, Carlsbad, CA, USA)) and transferred to Primaria tissue culture flasks (Becton Dickinson, Franklin Lakes, NJ, USA). The 1-h preplating applied four times finally allowed for achieving 60–70% myoblast purity [16 (link)]. Next, 100,000 cells were transferred to culture flasks. Isolated muscle cells of beef and dairy breeds were cultured in proliferation medium until 80% of confluence, which was changed every 48 h. Then, the cells were trypsinized and centrifuged, the supernatant was discarded, and the cell pellet was frozen for further analysis.