5 ethynyl 20 deoxyuridine edu

Cell proliferation was assessed using a CCK-8 Cell Counting kit (Beyotime, Shanghai, China). FBCs were seeded in a 96-well plate (2 × 10³ cells per well) and then cultured for 12, 24, 36, 48, 60, or 72 h before adding 10 μl of CCK-8 (5 mg/ml) to the culture medium in each well. After incubating for 2 h, absorbance at 450 nm was measured using a Thermo-max microplate (ThermoFisher, Massachusetts, United States) reader. All experiments involving each transfection were performed in triplicate. HPPCs were transferred to culture medium with 50 µM 5-ethynyl-20-deoxyuridine (Edu, Beyotime, Beijing, China) for 2 h at 37°C after 36 h transfection. Afterwards, cells were fixed in 4% paraformaldehyde for 15 min at room temperature (RT), and then permeabilized with 0.3% Triton X-100 for 10 min. To block non-specific binding, cells were incubated in blocking buffer (PBS containing 3% bovine serum albumin, 0.3% Triton X-100) for 1 h at room temperature. Next, the cells were incubated with a solution containing 10 mM Edu for 30 min in the dark. Nuclei were stained with 10 μg/ml 4, 6-diamidino-2-phenylindole (DAPI, Solarbio, Beijing, China) solution in the dark for 10 min. A Leica SP8 confocal microscope was used to capture three randomly selected fields to visualize the number of Edu-stained cells.