Anti cd1a

Fluorescein isothiocyanate, phycoerythrin, allophycocyanin, or Brilliant Violet 421 conjugated mAbs anti-CD1a, CD14, CD16, CD80, CD86, HLA-DR, CD83, CD206, CD1c, CD11b, DC-SIGN, CD4, CD3, CD39, CTLA-4, PD-1, Ki-67, IFN-γ, FoxP3, and CD25 were obtained from BD Pharmingen or BioLegend (Table 1). In all cases, isotype-matched control antibodies were used. Isotype staining and fluorescence minus one controls were performed using control DCs, DCs differentiated with PGE2 and with TGF-β (Figure S1 in Supplementary Material).
Analysis was performed by using a FACS Canto II flow cytometer (BD) and FlowJo X v10.0.7r2 software. The results are expressed as the mean fluorescence intensity (MFI), median fluorescence intensity, or as the percentage of positive cells.

Before and after differentiation, the cells were collected, washed and resuspended in Dulbecco’s phosphate-buffered saline (D-PBS, HyClone). To analyze surface antigens, anti-CD1a (BD Biosciences, San Jose, CA), anti-CD11c (BD Biosciences), anti-CD14 (Miltenyi Biotec Gmbh), anti-CD40 (BD Biosciences), anti-CD80 (Miltenyi Biotec Gmbh), anti-CD83 (Miltenyi Biotec Gmbh), anti-CD86 (Miltenyi Biotec Gmbh) and anti-CD209 (BD Biosciences) fluorescence monoclonal antibodies were used. Matched labeled isotypes were used as controls. The labeled cells were analyzed using flow cytometry (Accuri C6, BD Biosciences). The screening criterion of monocyte-derived DCs was defined as CD40⁺CD209⁺ cells in this study [55 (link), 56 (link)].