Annexin V-FITC/PI double staining for apoptosis evaluation by flow cytometry

SW13 cells at the logarithmic growth phase were placed on a 96-well plate. Each cell group was treated with drugs as previously mentioned. Twenty-four hours after drug treatment, the cells were dissolved by trypsinization and washed with cold PBS for three times. Then, the supernatant fluid was discarded, and binding buffer (400 µL) was added. Annexin V-fluorescein isothiocyanate/propidium iodide double staining was performed according to the manufacturer’s recommendations (Annexin V-FITC Apoptosis Kit). The excitation wavelength was 488 nm. Annexin V-fluorescein isothiocyanate (FITC) (6 µL) and propidium iodide (PI) (4 µL) were added to the cells, which were then cultured for 20 minutes. The apoptosis test was conducted in each group using the Cell Quest Software (BD, Franklin Lakes, NJ, USA) in the green and red fluorescence channels. Additionally, flow cytometry (FACScan, BD Biosciences) was used to analyze the results.