Trypsin

The heart tissue of newborn SD rats was cut into pieces after washing with precold PBS and 40 minutes of digestion with 2.0 g/L type II collagenase (Yeason, China) and 2.5 g/L trypsin (Yeason, China). Then the digest buffer was removed and the samples were cultured with Dulbecco's modified eagle medium (DMEM; Gibco, USA) containing 10% foetal bovine serum (FBS; Gibco, USA), followed by filtering with a 200-mesh copper mesh. The obtained cardiac fibroblasts were then cultivated in a complete medium (37°C, 5% CO₂). For the CF model, cardiac fibroblasts were treated with 100 nM angiotensin II for 24 h to induce an in vitro myocardial fibrosis model.

Each experimental group was established with three independent replicates. Cells were digested with 0.25% trypsin (Yeasen, Shanghai, China), precooled with 75% ethanol (Solarbio, Beijing, China), and fixed at 4 °C for 4 h, whereafter we added a PI staining solution (50 ug/mL, green, Solarbio, Beijing, China), then incubated the samples at 4 °C in the dark for 30 min. Using an Attune CytPix imaging flow cytometer (Thermo Fisher Scientist, Shanghai, China), detection was undertaken using standard procedures; the results were analyzed using cell cycle fitting software ModFit 3.0.

MC3T3-E1 cells were purchased from Cyagen Biosciences Inc. (M7-0201, Guangzhou, China). Cells were cultured with Minimum Essential Medium Alpha (α-MEM, HyClone, Utah, USA) containing 10% fetal bovine serum (FBS) (BioInd, Israel), 100 U/mL streptomycin, and 100 U/mL penicillin (BioInd, Israel) in an incubator (Heal Force, HF-240) at 37 °C and 5% CO₂. When the cell density reached 70–80%, it was digested with 0.25% trypsin (Yeasen) and then passaged.