Anti cd14 monoclonal antibody

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)].

RAW246.7 murine macrophages (ATCC, TIB71) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Corning, United States) supplemented with 10% fetal bovine serum, 1 mM glutamine, 100 U/μL penicillin and 100 μg/mL streptomycin (all from EuroClone, Italy). The cells were grown in tissue culture flasks or multiwell plates, at 37°C and 5% CO₂. Human monocyte-derived macrophages (HMDMs) were differentiated in vitro from monocytes isolated from the buffy coats of healthy donors, as previously described (Del Porto et al., 2011 (link)). Briefly, peripheral blood mononuclear cells were isolated by density gradient centrifugation (Lympholyte; Cedarlane, Hornby, CA, United States), and were selected with an anti-CD14 monoclonal antibody coupled to magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The CD14⁺ cells were differentiated for 7 days in Roswell Park Memorial Institute (RPMI) 1640 (Gibco-BRL, Invitrogen Corporation, Carlsbad, CA, United States) supplemented with 20% fetal bovine serum and 100 ng/mL recombinant macrophage colony stimulating factor (PeproTech Inch, Rocky Hill, NY, United States).

Primary monocyte-derived macrophages (MDMs) were prepared as previously described (17 (link)). Briefly, peripheral blood mononuclear cells (PBMCs) from healthy donors and CF patients were isolated by Ficoll density gradient, and monocytes were then positively sorted using anti-CD14 monoclonal antibodies conjugated to magnetic microbeads (Miltenyi Biotec), according to manufacturer’s instructions. Monocytes were then suspended in complete medium and incubated for a further 5 days in 96-well plates at a concentration of 10⁶ cells/ml in the presence of M-CSF (50 ng/ml, Miltenyi Biotec) to get differentiated macrophages.

Human blood monocytes from healthy volunteers were separated from peripheral blood mononuclear cells (PBMCs), by using anti-CD14 monoclonal antibodies conjugated to magnetic microbeads (Miltenyi Biotec), according to manufacturer's instructions. To obtain immature dendritic cells (iDc), cells were suspended in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-Glutamine and 5 μg/ml Gentamicin) and incubated for 5 days in 24-well plates at the concentration of 5 ×10⁵ cells/well in the presence of 20 ng/mL GM-CSF (Sigma-Aldrich) and 20 ng/mL IL-4 (Miltenyi Biotec). To obtain mature dendritic cells (mDC), iDCs were further stimulated for 18 h with 100 ng/ml lipopolysaccharides (Sigma-Aldrich).