Monocyte-Derived Macrophage Culture and Differentiation

MoMФ cultures were obtained from heparinized blood samples, as we previously published (22 (link)). In brief, leukocytes were cultured in RPMI-1640 supplemented with 10% foetal bovine serum (FBS), antibiotics (100 U/mL penicillin, and 100 μg/mL streptomycin) (complete RPMI, cRPMI), and recombinant human M-CSF (hM-CSF) (Thermo Fisher Scientific, Waltham, MA, USA) (final concentration 50 ng/mL), using Petri dishes (22 (link), 27 (link)). Cells were then seeded in 12-well plates (Greiner CELLSTAR, Sigma-Aldrich, Saint Louis, MO, USA) (1 × 10⁶ live moMФ per well) or 4-well chamber slides (Nunc Lab-Tek chamber slide system, Thermo Fisher Scientific) (3 × 10⁵ live moMФ per well). After seeding, macrophages were cultured in un-supplemented fresh cRPMI at 37°C 5% CO₂. 24 h later, moMФ were left untreated or differentiated into moM1, using recombinant porcine IFN-γ (Raybiotech Inc, Norcross, GA, USA) and LPS (lipopolysaccharide from Escherichia coli 0111:B4; Sigma-Aldrich), both at a concentration of 100 ng/mL (27 (link)). 24 h later, both moMФ and moM1 cultures were left untreated or exposed to diverse doses of goat mEVs (0.6, 60 μg) for 24 or 48h. In selected experiments (see 2.5), moMФ were instead exposed to scalar doses of goat milk EVs for 24h: 0.06, 0.6, 6, 60, 600 μg (protein weight).