*5.6. Human THP-1 Pre-Monocyte Differentiation into THP-1 Macrophages*

Human pre-monocytes were differentiated into macrophages using Phorbol 12- Myristate 13-Acetate (PMA, Sigma Aldrich, St. Louis, MO, USA), according to protocol described by Daigneault and colleagues (2010) [33]. Briefly, live cells were transferred to 24 culture wells plates containing 1 mL/well of supplemented RPMI medium containing PMA (100 ng/mL) at a density of 2 × 105 cells/well. The cell was incubated at 37 ◦C in an atmosphere containing 5% CO2 during 72 h. After the fourth day, PMA-containing medium was replaced by medium without PMA (2 mL/well), and the cells were kept at rest for 4 more days. At the end of the rest period, macrophage adhesion was visualized under a phase contrast microscope (Leica DM2500, Wetzlar, Germany) and photographed.
