2.4. Evaluation of Apoptosis

Apoptosis was measured using flow cytometry, using the Annexin V staining. Cells were washed once with PBS, detached with Accutase (Euroclone, Milan, Italy), resuspended in 100 mL of 1× Annexin-binding buffer at the concentration of 1 × 10⁶ cells/mL, stained with 5 mL of Annexin V FITC-conjugated (ImmunoTools, Friesoythe, Germany) and 1 mL of 100 mg/mL propidium iodide (PI) working solution and incubated at 4 °C in the dark condition for 15 min. Then, 400 mL of 1× Annexin Binding Buffer was added to each sample and cells were analyzed using flow cytometry (BD-FACS Canto) to find out the viability (annexin V and PI negative, Q3), early apoptosis (annexin V positive and PI negative, Q4), or late apoptosis (annexin V and PI positive, Q2). A minimum of 10,000 events were collected, as previously described [26].