Apc conjugated anti cd83

DC maturation was analyzed 24 hours post stimulation. Cells were stained with PE-conjugated anti-CD80 (1:25, 557227, BD Pharmingen), APC-conjugated anti-CD83 (1:25, 551073, BD Pharmingen) and FITC-conjugated anti-CD86 (1:25; 555657; BD Pharmingen). Cells were analyzed on a FACS Canto II (BD Biosciences).

MDDC were labelled (30 min at 4 °C) with different mix of FITC-conjugated anti-CD36, −CD86, −CD209, −CCR7 or -CD1a, PE-conjugated anti-B7H1, −CD80 or -CD54, APC-conjugated anti-CD83, −CD40 or -CD11c, PECy5-conjugated anti-HLA-DR and corresponding IgG isotype controls (BD Pharmingen™, BD Biosciences). After been washed and fixed in 0.25 % paraformaldehyde (PFA), MDDC were gated using FSC and SSC on a FACSCalibur flow cytometer with CellQuest software (BD biosciences). For intracellular staining, T-cells were incubated in RPMI 1640 supplemented with 10 % FCS plus brefeldin A (10 μg/ml) for 6 h at 37 °C. Cells were first labelled (30 min at 4 °C) with APC-Cy7-conjugated anti-CD45, Alexa Fluor 700-conjugated anti-CD4 and AmCyan-conjugated anti-CD8. After 15 min incubation, cells were fixed, permeabilized (Kit, BD Biosciences) and labelled with FITC-conjugated anti-IFN-γ, APC-conjugated anti-IL-17, PE-conjugated anti-IL-22 or the related isotype controls. Results are expressed as the difference between median fluorescence intensity (MFI) with the specific antibody and the isotype control (ΔMFI).