Cell Cycle Analysis of NPC Cells

This procedure was reported previously^{17 (link)}. Briefly, NPC cells were cultured in 6-well plastic plates at 2 × 10⁵ cells/well and treated with increased doses of β-elemene for 24 h. Afterwards, the cells were harvested, washed with phosphate-buffered saline (PBS), and resuspended in 500 μL of cold PBS and ethanol (1.5 mL) for 2 h at 4 °C. Afterwards, the fixed cells were incubated in 1 mL of 0.1% sodium citrate containing propidium iodide (PI) and RNase for 30 min at room temperature. The cells were washed and subjected to FACS Calibur flow cytometric analysis (FC500, Beckman Coulter, FL, USA), and the proportion of cells within the G0/G1, S, and G2/M phases of the cell cycle were analyzed using the MultiCycle AV DNA Analysis software (Phoenix Flow Systems, Inc. San Diego, CA, USA).