In Vitro Differentiation and Stimulation of Monocyte-Derived Macrophages

Mφ cultures were obtained from blood leukocytes, as previously described [21 (link)]. Leukocytes were cultured for 7 days in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 100 U/mL streptomycin, and 100 μg/mL penicillin (complete RPMI, cRPMI), and with 50 ng/mL of recombinant human M-CSF (hM-CSF) (Thermo Fisher Scientific, Waltham, MA, USA), using Petri dishes, as previously described [21 (link)]. MoMΦ were then harvested, washed, re-suspended in cRPMI and seeded in 12-well plates (Greiner CELLSTAR, Sigma-Aldrich, Saint Louis, MO, USA) (8–10 × 10⁵ live cells per well) or 8-well chamber slides (Nunc Lab-Tek chamber slide system, Sigma-Aldrich) (1 × 10⁵ live cells per well). Cells were incubated at 37 °C 5% CO₂ for further 24 h before stimulation. moMΦ were left untreated or stimulated with recombinant porcine IL-10 or TGF-β: culture medium was replaced with fresh cRPMI containing either IL-10 (20 ng/mL) or TGF-β (20 ng/mL) (both R&D Systems, Minneapolis, MN, USA). In defined experiments, culture medium was instead replaced with fresh cRPMI containing LPS (lipopolysaccharide from Escherichia coli 0111:B4; Sigma-Aldrich) (1 μg/mL) or recombinant porcine IL-4 (20 ng/mL) (R&D Systems), which were used as positive controls.