Cell Cycle and Apoptosis Assay

For cell cycle experiments, cells were collected in single-cell suspension and washed twice with precooled PBS. The cells were resuspended in pre-cooled 70% ethanol and fixed overnight at 4°C or kept at -20°C for long-term fixation. Subsequently, the cells were washed with 1 mL PBS and incubated at 4°C in the dark for 30 minutes with 500 mL PBS (including 5 μg/mL propidium iodide (PI), 100 μg/mL RNase A, and 0.2% Triton X-100). For cell apoptosis experiments, cells were collected in single-cell suspension and washed with precooled PBS and Binding Buffer, in turn. The cell suspension was prepared at 100 μL containing 1 x 10⁵ to 1 x 10⁶ cells. To each sample, 5 μL Annexin v-APC and 10 μL 7AAD were added and then incubated at room temperature in the dark for 15 minutes. Next, 400 μL precooled Binding Buffer was added to resuspend the mixture. Flow cytometry was used to detect apoptotic cells. Both results were analyzed using Flowjo software.