Cd1a fitc

For direct immunofluorescence staining of murine DC, the following phycoerythrin (PE)-conjugated rat anti-mouse antibodies were used (BD Biosciences Pharmingen): CD11c-PE (dilution 1:100), CD80-PE (dilution 1:600), CD86-PE (dilution 1:600), and CD40-PE (dilution 1:200). For direct cell staining, 50 μl of cell suspension was mixed with 50 μl of diluted antibody, and the mixture was incubated at 4 °C for 45 min. For indirect cell staining, 50 μl of cell suspension was incubated with an equal volume of unconjugated mouse anti-mouse I-A and MHC II (dilutions for both, 1:100) antibodies for 30 min at 4 °C, washed, and further incubated at 4 °C for 45 min with AlexaFluor 488-conjugated rat anti-mouse antibody (dilution 1:400; BD Biosciences Pharmingen).
Human dendritic cells were labeled directly with monoclonal antibodies against CD11c-APC (dilution 1:100), HLA-DR-FITC (dilution 1:50), CD83-APC (dilution 1:50), CD86-PE (dilution 1:100), CD1a-FITC (dilution 1:50) (BD Biosciences Pharmingen). For cell staining, 50 μl of cell suspension was mixed with 50 μl of diluted antibody, and the mixture was incubated at 4 °C for 45 min. Appropriate isotype antibodies were used as controls to determine non-specific binding. Cells were analyzed using a FACSCalibur flow cytometer (Becton-Dickinson, USA).

The following antibodies were used: CD1a-FITC (BD Pharmingen, San Diego, CA, USA), CD1a-APC, CD3-PE (both BD Bioscience, San Jose, CA, USA), CD4-PerCP (BD Pharmingen), CD4-Alexa488 (Biolegend, San Diego, CA, USA), CCR5-APC (CD195) (BD Pharmingen), CXCR4-PerCP (R&D systems, Minneapolis, MN, USA), Langerin-PE (CD207), p24-PE (both Beckman Coulter, Fullerton, CA, USA).

Isolated CD103⁺ DCs (1 × 10⁶) were subjected to flow cytometric analysis. Mouse-anti-rat CD54-Alexa Fluor^®488 (R&D Systems), CD86-phycoerythrin (PE)-Vio770 (Miltenyi Biotec), CD80-PE (eBioscience, San Diego, CA, USA), CD11b/c-allophycocyanin (APC; Biolegend, San Diego, CA, USA), RT1B (MHC- II)-FITC (BD Biosciences, San Jose, CA, USA), CD1d-APC (eBioscience), and the respective matched isotype controls were employed.
The following markers were used for the staining of imDCs (1 × 10⁶): mouse-anti-human CD80-FITC, CD54-PE, CD86-peridinin chlorophyll (PerCP)-Cy™5.5, MHC-II-APC, CD1a-FITC, CD83-PE, CD11c-PerCP-Cy™5.5, and the respective matched isotype controls (BD Biosciences). All samples were washed, resuspended in 2% PFA, and subjected to flow cytometric analysis (Accuri C6; BD Biosciences).