Cell apoptosis assay

HUVECs apoptosis was quantified by an Annexin V / PI detection kit (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions and analyzed by flow cytometry (BD Biosciences, San Jose, CA, USA). Cells were seeded in sterile 6-well plates at a density of 3×10⁵ cells/ml and incubated with TFA at different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml). After incubation of 72 h, the harvested cells were suspended in binding buffer at a density of 1×10⁶ cells / ml. Then, 5 μl of Annexin V- Fluorescein isothiocyanate (FITC) and 5 μl of PI were added to 100 μl of cell resuspension solution and the mixture was placed in the dark at RT for 15 min. 400 μl of binding buffer was added and the cells were analyzed by FACS. At least 10,000 cells per sample were analyzed.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL; Vazyme, Nanjing, Jiangsu, China) was also performed to observe the effect of TFA on HUVECs apoptosis. HUVECs were seeded on sterile coverslips in 6-well culture plates at a density of 3×10⁵ cells/ml to allow attachment. When cells reached 60% confluence, the medium was removed. Then, HUVECs were treated with TFA at different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml). Afterwards, the cells were fixed with 4% paraformaldehyde for 25 min at 4°C. Fixative was removed and coverslips were washed twice with PBS. Then sufficient volume of the permeabilization reagent was added to completely cover the coverslips. After washed twice with PBS, the coverslips were incubated with 1× Equilibration Buffer for 15 min at room temperature. 100 μL of terminal deoxynucleotidyl transferase (TdT) reaction cocktail was added to each coverslip and the solution was allowed to spread completely over the surface in a humidified chamber for 60 min at 37°C. Each coverslip was washed twice with PBS and then incubated with 4',6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. The percentage of TUNEL positive cells was calculated under a fluorescent microscope (Olympus, Tokyo, Japan) at 200× magnification.