Cell Cycle Analysis of A375/B16 Cells

A375/B16 cells were treated with different concentrations of cinobufagin for 24 h, and then collected by digestion and made into cell suspensions. The cells were fixed in pre-cooled 70% ethanol solution at 4°C for 2 h and then centrifuged at 800× g in a low temperature centrifuge for 5 min. The ethanol solution was discarded and the cells were washed twice with PBS. Then they were precipitated and suspended in 500 μL phosphate buffer containing 0.02 mg/mL propidium iodide (PI) (Cat no. ST512; Beyotime, Shanghai, China) and 0.1 mg/mL Ribonuclease A (Cat no. ST576; Beyotime, Shanghai, China). After 30 min incubation in the dark at 37°C, the cells were precipitated and suspended in phosphate buffer solution. The Epics XL Flow Cytometer (Beckman Coulter, Inc., Brea, CA, USA) was used to detect fluorescence of the PI-DNA complex, and Winmdi 2.8 software (Scripps Research, La Jolla, CA, USA) was used to analyze the cell distribution at different stages of the cell cycle (25 (link)).