*5.7. CD11b Expression in Differentiated THP-1 Macrophages*

The CD11b expression by differentiated THP-1 macrophages was evaluated by flow cytometry, according to the literature [32], with some modifications, as described below. THP-1 monocytes were cultured in 75 cm<sup>3</sup> culture flasks until reaching a density of <sup>8</sup> × <sup>10</sup><sup>5</sup> cells/mL, according to what is described in 5.5. At this time, the culture medium was replaced with medium containing 100 ng/mL of PMA, and differentiation was conducted as described in 5.6. The adherent macrophages were carefully detached with cell scraper and centrifuged (260× *g*) for 10 min at 10 ◦C. Contents of viable cells were determined by Trypan blue exclusion, and the cell pellet was resuspended in FACS buffer (PBS with 1% BSA and 0.01% sodium azide) at the density of 20 × 106 cells/mL. Cells were transferred to 96 round-well-bottom microplates (50 μL/well) and were incubated protected from light for 30 min at 4 ◦C with anti-CD11b PE-labelled antibody (IgG1κ PE, Clone VIM 12, BD, Becton Dickinson, San Jose, CA, USA) or with isotypic control (IgG1κ PE, BD) diluted at 1:5 (*v*/*v*) in FACS buffer. After incubation, the cell suspensions were centrifuged (305× *g*) for 5 min at 4 ◦C, washed three times with FACS buffer and resuspended in 300 μL/well of FACS buffer with 1% paraformaldehyde. The data acquisition was performed in flow cytometer (FACS Aria III, Becton Dickinson, San Jose, CA, USA). In parallel, the CD11b expression was also evaluated in THP-1 monocytes as control.
