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.