Clec10a

The purity of MDDCs and mDCs were verified by flow cytometry, and was shown to be ~90% pure for CD11c and CD1c respectively. The DCs were stained for C-type lectins using anti-CD205, -CD206, -CD207, -CD209, -CLEC4A, -CLEC9A, -CLEC10A, -CLEC12A (Biolegend) and -CD303 (Miltenyi Biotec) antibodies conjugated to PE. FACS data was acquired on BD FACS Calibur (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star).

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.).