Annexin 5 allophycocyanin 5 apc

When A375 and SK-MEL-28 cells grew to a coverage rate of 70%, the cells were collected and washed once with 4 °C pre-cooled PBS. The cells were fixed with 70% ethanol pre-cooled at 4 °C for at least 1 h. After washing with PBS, the cells were resuspended with the proportioned cell staining solution, and then a flow cytometer (Guava easyCyte HT, Millipore) was used to detect the cell cycle distribution. Cell staining solution configuration ratio: 40× propidine iodide (PI) (2 mg/ml, P4170, Sigma, MO, USA): 100× RNase (10 mg/ml, 2158-1, TakaRa Biomedical Technology Co., Ltd., Beijing, China): 1× PBS = 25:10:1000. When A375 and SK-MEL-28 cells grew to a coverage rate of 70%, the cells were collected and washed with D-Hanks and 1× binding buffer pre-cooled at 4 °C. The cells were resuspended in 200 μl of 1× binding buffer. 10 μl of Annexin V-allophycocyanin (V-APC) (eBioscience, CA, USA) was added to the cell suspension and incubated for 15 min at room temperature in the dark. Flow cytometer (Guava easyCyte HT, Millipore) was employed to detect cell apoptosis. This experiment was repeated three times independently.