Cd1a clone hi149

Monocytes were isolated from cryopreserved PBMCs as described above and cultured for 5 d in complete RPMI with 10% FBS at 10⁶ cells/ml in the presence of 100 ng/ml M-CSF, 40 ng/ml of IL-4 (Miltenyi Biotec), and 5 ng/ml TNF (R&D Systems). After culture, cells exhibiting the macrophage or DC phenotype were quantified using flow cytometry staining for CD16 (clone 3G8; BioLegend) or CD1a (clone HI149; BioLegend) markers, respectively. CD163 (clone GHI/61; BioLegend) and viability dye (DAPI) were also included in the staining panel, and the staining buffer was composed of PBS with 0.5% human serum and 2 mM EDTA. For morphological analysis, cells were cytospun, stained with May–Grünwald and Giemsa solutions (Sigma-Aldrich), and imaged using a CFW-1308C color digital camera (Scion Corporation) on a Leica DM 4000 B microscope (Leica Microsystems).

Dead cells were excluded by the fixable viability dye eFluor 780 (eBioscience) and FcR-dependent staining was blocked with human FcR blocking reagent (Miltenyi). Cells were stained with fluorophore-conjugated antibodies against CD45 (clone HI30, BioLegend), CD14 (clone HCD14, BioLegend), HLA-DR (clone L242, BioLegend), CD1a (clone HI149, BioLegend), human Langerin (clone MB22-9F5, Miltenyi) for either 30 min or 15 min on ice. Experiments were conducted with an Attune NxT flow cytometer (ThermoScientific) or FACS Canto II (BD Biosciences) and analysed in FlowJo software.