Apoptosis Evaluation of Treated HNPCs

The apoptosis of treated HNPCs was evaluated using flow cytometry [26 (link)]. Prior to this evaluation, the Annexin V-FITC/Propidium Iodide Apoptosis Detection Kit (C1062M) was purchased from Beyotime (Shanghai, China), and treated HNPCs were collected and digested with Trypsin solution. Briefly, treated HNPCs were resuspended with PBS and the cell concentration was adjusted, after which cells (5 × 10⁴) were centrifuged (1000 × g) using a centrifuge (E2615, Beyotime, Shanghai, China) for 5 min. Afterward, cells were added with 195 μL AnnexinV–FITC conjugated solution, 5 μL of Annexin V-FITC solution and 10 μL of Propidium Iodide solution, followed by 15-min incubation at room temperature in the dark. Ultimately, the apoptosis of treated HNPCs was analyzed using flow cytometer (CytoFLEX, Beckman Coulter, Inc., Kraemer Boulevard Brea, California, USA) and the Kaluza C software (v. 1.1.2, Beckman Coulter, Indianapolis, Indiana, USA).