Isolation and Culture of Primary Cardiac Fibroblasts

Primary CFs were isolated from the left ventricles of neonatal SD rats following the protocol described in a previous study (12 (link)). After having been harvested from 2-day-old SD rats, the hearts were minced and digested using type II collagenase (120 U/ml; Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). The dissociated cells were collected by centrifugation (200 × g for 8 min at room temperature) and further cultured in MEM medium (Hyclone™) supplemented with fetal bovine serum (FBS; 10%, Hyclone™) (both from GE Healthcare Life Sciences, Shanghai, China) and penicillin-streptomycin antibiotic mix (Sigma-Aldrich; Merck KGaA). Cells were incubated in a humidified incubator under conditions of 95% fresh air and 5% CO₂ at 37°C. The adherent cells were cultured to reach over 80% confluence. In excess of 95% of the collected cells were vimentin-positive/α-actin-negative. Cells from passages 2–3 were used. Table II shows the details of grouping, and treatments of the isolated cells.