Neonatal Cardiac Fibroblast Isolation and Choline-Induced TGF-β1 Treatment

All animal care and laboratory procedures of this study were approved by the Ethical Committee of Harbin Medical University and all animal were obtained from the Experimental Animal Center of Second Affiliated Hospital of Harbin Medical University, China. Neonatal mouse (1–3 days old) hearts were prepared for finely minced and placed together in 0.25% trypsin to get single cell suspension. After the cells were filtered and centrifuged (1000 rpm/min, 5 min) and then resuspended in DMEM (HyClone, USA) containing 10% fetal bovine serum and 5% penicillin/streptomycin. Finally, the cells were plated into Petri dishes (60 mm) or other specifications of culture plate and cultured under a condition of 5% CO₂ and 95% air at 37 °C for 1.5 h. Then, cardiac fibroblasts preferential attached the bottom of petri dishes and new DMEM containing 10% fetal bovine serum and 5% penicillin/streptomycin was replaced in the Petri dishes to culture primary cardiac fibroblasts. The cardiac fibroblasts were pre-stimulated with choline (5 mM) for 1 h and then incubated with recombinant human TGF-β1 (20 ng/mL, Sigma-Aldrich Co., LLC, USA) for 48 h. Choline (1 mM) treatment was operate as describe previously [19 (link)]. The cells were used for the experiments.