Apoptosis Detection by Flow Cytometry

The detection of phosphatidylserine externalization was performed using an Annexin V Apoptosis Detection Kit (K101-100 BioVision CA, USA), made up of annexin V–fluorescein isothiocyanate (AnV–FITC) and propidium iodide–phycoerythrin (PI–PE), which are able to differentiate viable from necrotic and apoptotic cells. The aliquots of experimental samples were washed with PBS (Euroclone, Milan, Italy), centrifuged, and suspended in 500 μl of Annexin binding buffer to obtain a cell count of approximately 1 × 10⁵. Five microliters of AnV–FITC and 5 μl of PI–PE (50 μg/ml) were added to each cell suspension. The samples were incubated at RT for 5 min in the dark and then analyzed by a flow cytometer. Flow cytometry analysis was performed by plotting green fluorescence (FL1)/AnV–FITC vs. red fluorescence (FL2)/PI–PE positive cells. The combination of AnV and PI allows the discrimination of four cell categories: viable cells (AnV−/PI−), early apoptotic cells (AnV+/PI−), late apoptotic cells (AnV+/PI+), and necrotic cells (AnV−/PI+). The sum of apoptotic cells was also calculated. Flow cytometry data acquisition was performed on a FACSscan Calibur (Becton Dickinson, Milan, Italy) equipped with 488- and 633-nm lasers and running CellQuest Software (Becton Dickinson, CA, USA). Ten thousand events were collected for each sample (43 (link)).