2.12. Apoptosis assay
Apoptosis was evaluated by flow cytometry by measuring the levels of Annexin V (A) and Propidium Iodide (PI). Briefly, cells (2 x 104 cells/well) were seeded in 6-well plates and incubated with Dulbecco’s Modified Eagle Medium/Ham F-12 (DMEM/F12) containing 10% Foetal Bovine Serum (FBS), 2% GlutaMAX 100X and 1% antibiotic-antimycotic (Thermo Scientific, Spain) for 24 h, at 37 ºC, 5%CO2. ON cells were detached with 0.25% trypsin-EDTA (GibcoBRL, USA), and incubated with anti-Annexin V (PB-V450, #560506, BD) and PI (#556463, BD) at 4ºC, 30 min. Fluorescence was measured with a Cytoflex cytometer (Beckman Coulter) and the CytoExpert software. Finally, data were analysed with FlowJo v 10.4 software.