Mouse serum

For staining, cells were first detached with trypsin (Life Technologie) and washed twice in PBS containing 2% FCS (Biowest) and 0.1% sodium azide (NaN3, Sigma-Aldrich). Next, antigen nonspecific binding was prevented by prior incubation of cells with 10% mouse serum (Fitzgerald). Cells were next incubated with combinations of pacific blue, fluorescein isothiocyanate (FITC), and allophycocyanin (APC) –conjugated mouse anti–human antibodies. The following monoclonal antibodies were used: mouse-anti-human CD3 (Clone UCHT1, Biolegend), mouse-anti-human CD19 (Clone HIB19, BD Biosciences), mouse-anti-human CD56 (Clone B159, BD Biosciences), and mouse-anti-human HLA-ABC (Clone W6/32, Biolegend). Cells were acquired on FACSCanto II and analyzed using FACS Diva Version 6.13 (BD Bioscience) or FlowJo version 7.6.5 software. Data were analyzed using GraphPad Prism 5.

To measure viability, after moDC differentiation, stimulation or transfection, cells were first incubated with Fixable Viability Dye eFluor780 (eBioscience) in PBS to exclude dead cells. Non-specific Ab binding was prevented by treating the cells and were further treated with 10% (v/v) mouse serum (Fitzgerald). For phenotypic analysis, cells were stained with the following anti-human fluorochrome-conjugated mAbs: CD14 (1:120 dilution; clone M5E2), CD86 (1:80 dilution; clone IT2.2) and CLEC10A (1:50 dilution; H037G3) obtained from BioLegend, CD1a (1:70 dilution; clone HI149) and LAMP1/ CD107a (1:50 dilution; clone H4A3) obtained from BD, or the isotype control-matched Ab. Cells were acquired on the LSR Fortessa (BD) and data was analyzed using the FlowJo software (version 7.6.5; Tree Star. Inc.).