Bb 4101

The effects of serum starvation and TUDCA-treatment on the cell cycle and apoptosis of nuclear donor cells were examined according to the experimental designs. Cells were digested by 0.25% trypsin in D-Hanks solution for 3 min at 37°C, rinsed 2 times with chilled PBS, fixed in 75% ethanol by freezing for 1 h at -20°C, rinsed once with chilled PBS again, resuspended with 400 μL cold PBS containing 20 μL of RNase A, incubated at 37°C for 30 min, filtered through a 400-mm mesh, mixed with 400 μL of propidium iodide (100 mg/mL) staining solution in the dark, incubated for 1 h at 4°C, and finally examined by flow cytometry (Becton-Dickinson, Oxford, UK). For cell apoptosis analysis, cells were collected by trypsin digestion. After washing 3 times with cold PBS, 400 μL of annexin V (BestBio, BB-4101) and 5 μL of annexin V-EGFP staining medium (BestBio, BB-4101) were added. The cell mixture was mixed slightly, incubated in the dark for 15 min at 4°C, supplemented with 10 μL of propidium iodide (10 mg/mL), incubated for 5 min, and finally examined by flow cytometry (Becton-Dickinson).

To analyze the cell cycle, (5–10) × 10⁶ cells were harvested, washed twice with cold PBS, and fixed in 70–90% ethanol at − 20 °C overnight. Post-fixation, cells were washed and resuspended in PBS, treated with 20 μL of RNase A for 30 min at 37 °C, and stained with 400 μL of propidium iodide (PI) solution (Bestbio, BB-4101, China) for 30 min in the dark at 4 °C. The stained cells were then subjected to flow cytometric analysis to determine the distribution of cells across different phases of the cell cycle.

Heart tissue samples were treated as described previously [18 ]. CD31 and TUNEL assay (Sigma, USA) double immunofluorescence staining was performed to measure CMECs apoptosis in heart tissue. Fluorescent staining was visualized using a confocal laser scanning microscope (Olympus FV3000, Japan). For cell samples, a biological apoptosis test kit (BestBio, China, BB-4101) was used for detection. The pancreatic enzyme without EDTA was used to digest the CMECs, and the treated cells were collected with the medium into a 15 ml centrifuge tube. The tubes were centrifuged at 1000 rpm for 5 min and washed twice in precooled PBS. Binding Buffer binding solution (400 µl) was added and mixed, and 10 µl Annexin V-FITC was added for incubation in the dark at RT for 15 min. 7 µl PI dye was added, gently mixed, and incubated in the dark at 4℃ for 5 min. Flow cytometry was used for detection and analysis.