Isolation and Culture of Neonatal Mouse Cardiomyocytes

Hearts were isolated from 40 decapitated 1-to 3-day-old neonatal wild type mice with the atria dissected away were minced and digested with 108 U/ml collagenase type II (Worthington) and 0.9 mg/ml pancreatin (Life Technologies, Grand Island, NY) to obtain free cells. Myocytes were plated on gelatin-coated dishes overnight in DMEM/medium 199 (4:1) supplemented with 10% horse serum, 5% fetal calf serum, 2 mM l-glutamine (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (EuroClone) at a density of 1 × 10⁵ cells/cm². The next day cells were rinsed three times and the plating medium was replaced with serum-free medium consisting only of DMEM/medium 199 (4:1), 2 µM l-glutamine (Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (EuroClone); 10 µM cytosine-ß-d-furanoarabinoside was added to stop proliferation of non-cardiomyocytes and cultures contained >95% cardiac myocytes^{44 (link)}. Cells were serum starved for 6 h before starting the experiments of co-culture with macrophages.