4.4. Apoptosis Analysis by Flow Cytometry

Detection of apoptosis was performed using the Annexin V/PI kit (#640932, Biolegend, San Diego, CA, USA), according to the manufacturer’s protocols. After 48 h of metformin incubation (10 mM) or everolimus (10 nM), alone or in combination, cells were double-stained with fluorochrome-labeled annexin V and red-fluorescent propidium iodide dye. The percentage of apoptotic cells was determined by flow cytometry using Fluorescence-activated cell sorting _(FACS)LSRFortessa (BD Biosciences, Franklin Lakes, NJ, USA). The results were analyzed using FACSDiva software (BD Biosciences).