Flow Cytometry Analysis

Cell apoptosis was detected using an Annexin V-FITC apoptosis kit and following the provided procedure (Beyotime, Shanghai, China). After pretreatment with periplocymarin for 24 h, the cells were treated with 5 μM of DOX for 12 h. Then they were harvested using 0.25% trypsin and washed twice with cold phosphate-buffered saline (PBS). The harvested cells were centrifuged at 2,000 rpm/min for 5 min and the cells were resuspended with 1 × binding buffer to a density of 1 × 10⁵ cells/ml. Afterward, 100 μl of each sample was stained with an Annexin V-FITC staining solution in a 1.5 ml Eppendorf tube at room temperature and avoided light for 15 min. Then, 400 μl of 1 × binding buffer was added into the tubes. The apoptotic cell population was then quantified using a flow cytometer (BD FACSVerse, Becton, Dickinson and Company, United States) and Cell Quest Research Software (FlowJo_V10, Becton, Dickinson and Company, United States).