Flow cytometric analysis for apoptosis

Two days following cell injection, 0.25% trypsin was employed for cell harvest (2×10⁶). Phosphate-buffered saline (PBS) was pre-cooled at 4°C and used to wash the collected cells. In accordance with the instructions of the Annexin V-FITC Apoptosis Detection Kit (Beyotime Institute of Biotechnology), the obtained cells were resuspended in 195 µl Annexin V-FITC, followed by mixing with 5 µl Annexin V-FITC and 10 µl propidium iodide. Subsequently, the cells were cultured at 25°C for 15 min and continually maintained in darkness. The early + late apoptotic cells were detected by a flow cytometer (FACScan™; BD Biosciences) and analysed with CellQuest software v.2.9 (BD Biosciences).