Monocyte-derived Macrophage Isolation and Characterization

PBMCs were isolated from buffy coats using the standard LymphoprepTM (Accurate Chemical-Scientific, Westbury, NY, USA) gradient centrifugation. For the enrichment of monocytes, PBMCs were cultured for 2 h in a Corning^® 100 mm TC-treated culture dish (Corning, New York, NY, USA) and allowed to adhere. Non-adherent cells were removed by washing, and adhered cells were recovered using a cell scraper, and positive selection was performed using magnetic microbeads coated with an anti-CD14 monoclonal antibody (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of the CD14⁺ cell fraction was analyzed by flow cytometry using anti-human CD14, CD2, and CD19 monoclonal antibodies (mAbs) procured from BioLegend (San Diego, CA, USA). The enrichment efficiency of the CD14⁺ cell fraction was routinely >96%, as analyzed by flow cytometry (Figure S1). To obtain MDMs, CD14+ cells were cultured in Costar^® 6-well plates at a density of 2 × 10⁶ cells/well (Corning, New York, NY, USA) in RPMI-1640 culture medium (GIBCO, Grand Island, NY, USA), supplemented with 2 mM L-glutamine (GIBCO), 100 µg/mL streptomycin, 100 IU/mL penicillin, and 10% fetal bovine serum (FBS, GIBCO) for 7 days at 37 °C, in an atmosphere of 5% CO₂. After that, MDMs were recovered and characterized by immunophenotyping based on the expression of differentiation molecules, as previously reported [7 (link),30 (link)].