Annexin V-FITC and PI Apoptosis Assay

For the apoptosis assay, Annexin V–fluorescein isothiocyanate (FITC) detection kit (BD Biosciences, Erembodegem, Belgium) was used according to the manufacturer’s introduction. The cells were collected, washed twice with PBS, resuspended in Annexin V–FITC and propidium iodide (PI), and stained in the dark for 15 min at room temperature [29 (link)]. Subsequently, the cell apoptosis rate was analyzed using flow cytometry (fluorescence-activated cell sorting, BD Biosciences). For cell cycle distribution analysis, the cells were harvested and fixed in 70% ice-cold ethanol overnight. Then, the cells were centrifuged and resuspended in PBS containing PI (BD Biosciences) and RNase (100 μg/mL) as well as Triton X-100 (0.2%) for 30 min. Finally, flow cytometry was used to analyze cell cycle distribution [29 (link)]. The tests were performed three times for each sample.