Clone 19

Macrophages from PB-purified monocytes were stained with the anti-human MHC-II conjugated to PE (clone L243, BD) or anti-human CD86 conjugated to PE (clone IT2.2; BD) or anti-human CD64 conjugated to FITC (clone 10.1; BD) or anti-human CD206 conjugated to PE (clone 19.2, BD) or anti-human DC-SIGN conjugated to FITC (clone DCN46, BD) or anti-human CD163 conjugated to PerCP (clone GHI61, Biolegend) or anti-mouse MHC-II conjugated to FITC (I-A/I-E, clone M5/114.15.2, e-Bioscience) or the corresponding isotype control antibodies and then evaluated by flow cytometry at different times post-stimulation. When possible, cells were washed and incubated with 7-AAD (BD Biosciences) for 10 minutes on ice and in the dark. The expression of the different surface markers was evaluated within the viable cell population (7-AAD negative). After labelling, the cells were analysed on a FACSCalibur flow cytometer (BD Biosciences) or Partec CyFlow (LabSystems) and the data were processed with the FlowJo 7.6.2 or vX.0.7 applications (FlowJo, LLC). Data was normalized to untreated cells, i.e., in each set of experiments, for each marker, the MFI of treatments (from the % of positive cells for each marker) was divided by MFI of media condition (from the % of positive cells for each marker).

For surface staining moDC were incubated with FITC-conjugated mouse anti-CD40 mAB (10 µl/stain, Clone B-B20, Cat ab27281, abcam), APC-conjuagted mouse anti-CD83 mAB (10 µl/stain, Clone HB15e, Cat 551073, BD Pharmingen) and PE-conjugated mouse anti-CD14 mAB (10 µl/stain, Clone MΦP9, Cat 345785, BD Pharmingen) or isotype controls (5 µl/stain; mouse; monoclonal; FITC-IgG1, Cat ABIN118618, Antibodies-Online, Aachen, Germany; APC-IgG1, Cat 555751, BD; PE-IgG2b, Cat 555743, BD) for 15 min at 4°C and fixed in 1% PFA. MΦ were fixed and permeabilized using the Cytofix/Cytosperm kit (BD Pharmingen) and were then incubated with FITC-conjugated mouse anit-CD68 mAB (5 µl/stain, Clone Y1/82A, Cat 562117, BD Pharmingen), PE-conjugated mouse anit-CD163 mAB (10 µl/stain, Clone GHI/61, Cat 556018, BD Pharmingen), PerCP-conjugated mouse anti-HLA-DR mAB (10 µl/stain, Clone L243, Cat 347402, BD), and APC-conjugated mouse anti-CD206 mAB (5 µl/stain, Clone 19.2, Cat 550889, BD Pharmingen) or isotype controls (5 µl/stain; mouse; monoclonal; FITC-IgG2b, Cat 555057, BD; PE-IgG1, Cat 555749, BD; PerCP-IgG2a, Cat 349054, BD; APC-IgG1, Cat 555751, BD) for 30 min at 4°C. Flow cytometric analysis was performed on a FACSCanto II flow cytometer using the FACSDiva software v6.1.3 (BD Pharmingen). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).