Annexin V-PI Assay for Apoptosis

For identifying cells actively undergoing apoptosis, a double staining with Annexin V and PI was performed using Alexa Fluor^®488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States) as previously described (Scicchitano et al., 2023 (link)). After staining, cells were incubated at room temperature for 15 min in the dark. Each tube was diluted with 400 μL of Annexin Binding Buffer and then cells were analyzed by flow cytometry using the FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Inc.). Three independent experiments were conducted.