2.1. Cell Culture

Isolation and culture of chicken embryo fibroblasts and myocardial cells followed methods previously described [23, 24] with some modifications. Briefly, White Leghorn eggs were obtained from Beijing Merial Vital Laboratory Animal Technology (Beijing, China). At embryonic day 11 (E11), embryos were removed and decapitated in a Petri dish filled with Medium 199/EBSS (HyClone, Logan, Utah, USA) supplemented with 3% fetal bovine serum (FBS, Gibco, Grand Island, New York, USA). Ventricular tissues and torso of chicken embryo were isolated for the preparations of myocardial cells and CEF and treated with 0.05% trypsin-EDTA to obtain a cell suspension as described [14, 25, 26], respectively. Specifically, the cells of CEF and myocardial cells were, respectively, incubated at 8 × 10⁵ per well in 24-well plates in growth medium (Medium 199/EBSS containing 10% FBS) at 37°C under a 5% CO₂ atmosphere. Cultures were washed three times at 8, 24, and 48 h to remove dead and dying cells. The serum concentration in the medium was then changed from growth (10%) to maintenance (2%) conditions.

293T cells at low passage were incubated at 1 × 10⁵ cells/well in 24-well plates with Dulbecco's modified Eagle medium (DMEM) (HyClone, Logan, Utah, USA) containing 10% FBS at 37°C under a 5% CO₂ atmosphere. After plating, the serum concentration was decreased from growth (10%) to maintenance (2%) conditions.