3.5. Annexin V-FITC/PI Staining

Annexin V/ PI is a commonly used approach for studying apoptotic cells. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability. The fluorescence of AnnexinV-FITC/PI assay was read using flow cytometry. HUVEC cells were seeded on a 12-well plate at a density of 2.5 ×10⁴ and grown for 48 h before treatments. During the last 24 h cells were starved in the same EBM-2 basal medium containing 0.2% fetal bovine serum without EC (endothelial cell) supplements. Cells were then exposed to 5 µM peptide for 72 h. When required, 10 µM CuSO₄ was used in combination with peptides. Five hours of incubation in the presence of 1 µM STS (staurosporine) was used as a positive control for cell death. Cells were harvested and stained with Annexin V-FITC (Fluorescein isothiocyanate) and propidium iodide using an annexin V-FITC apoptosis detection kit (Life technologies, Monza (MB), Italy), following the manufacturer’s instructions. Briefly, cells were pelleted and resuspended in 800 µL PBS buffer containing 20 µL of annexin V, 20 µL of propidium iodide, and PI (Annexin V/Dead Cell Apoptosis Kit (Invitrogen), and kept for 10 min at 25 °C in the dark. Samples were analyzed within 1 h by flow cytometry using a CyFlow^® ML (Sysmex Partec Milan-Italy). The fluorescent scatter plots indicated three types of cell populations: viable (Annexin V-FITC−/PI−), dead (Annexin V-FITC + /PI+), and apoptotic (Annexin V-FITC + /PI−). Quadrant analysis (FlowMax software -Partec) was performed on the gated fluorescent scatter plot to examine the percentage of live, dead, and apoptotic cell populations. Experiments were repeated twice in triplicate.