*2.1. THP-1 Macrophage Differentiation*

Human non-adherent pre-monocytes of the THP-1 lineage were cultured in supplemented RPMI medium at a density of 2–8 × <sup>10</sup><sup>5</sup> cells/mL (Supplementary Figure S1A). In these conditions, the cells grew in clumps and showed a high rate of proliferation, doubling the number of cells within 2–3 days. When the cells reached high concentrations, not exceeding 1 × <sup>10</sup><sup>6</sup> cells/mL, live cells were differentiated in macrophages using PMA (Supplementary Figure S1B). In contrast to monocytes, differentiated macrophages become adherent and relatively larger than undifferentiated cells, stopping proliferation and spreading.

To confirm the differentiation, the expression of CD11b was evaluated by flow cytometry. CD11b, which together with CD18 forms the inactive C3b (iC3b) receptor, known as CR3 receptor, has been reported to be induced during differentiation of monocytes into macrophages. CR3 has important functions both as an adhesion molecule and a membrane receptor mediating recognition of different ligands [31–33]. As showed in Supplementary Figure S1C, only differentiated macrophages expressed CD11b, confirming the differentiation of these cells for further treatments.
