Kga1030

To conduct cell cycle detection, the two cells were treated with 1.2 mL of pre-cooled 100% ethanol, resulting in a final concentration of 75%. Subsequently, the cells were fixed by overnight incubation at 4℃. After the cells were washed with PBS, they were centrifuged. Then, they were mixed with 150 µL of propidium iodide (PI, MB2920, Meilune, China) and stained for 30 min at 4℃. Different stages of the cell cycle have different DNA content, and PI can label DNA to determine which cycle the cell is in. Detection of stained cells with a flow cytometer (A00-1-1102, Beckman, USA), PI was excited by a 488 nm argon laser and detected through a 630 nm bandpass filter, collecting around 15,000 cells in FSC/SSC dot plots, and analyzing the percentage of cells in each cell cycle phase on the PI fluorescence histogram to determine positive cell staining. For apoptosis detection, SK-Hep-1 and HCC-LM3 cells were treated with trypsin (AWC0232, Abiowell, China). Following digestion, after washing the cells with PBS, approximately 3.2 × 10⁵ cells were collected for further analysis. Following the instruction provided by the apoptosis detection kit (KGA1030, KeyGEN BioTECH, Nanjing, China), the cell suspension was treated with Annexin V-FITC (5 µL) and PI (5 µL) successively. The mixture was then incubated at room temperature for 10 min in the absence of light before further analysis.

Apoptosis was detected according to the instructions of the apoptosis kit (KGA1030, KeyGEN BioTECH). Briefly, cells were digested with EDTA-free trypsin and washed with PBS to a concentration of 3.2 × 10⁵ cells. Cells were suspended by adding 500 µL of Binding buffer. Subsequently, the percentage of apoptotic cells was assessed by flow cytometry (A00-1-1102, Beckman).