Hla dq fitc

For phenotypic analysis, 100,000 cells at the third passage were incubated with human monoclonal antibodies labeled with either fluroisothiocyanate (FITC) or phycoerythrin (PE) (CD11b FITC, CD19 PE, CD34 FITC, CD44 PE, CD45 FITC, CD73 PE, CD90 FITC, CD105 PE, HLA-DQ FITC, and HLA-DR PE; BD, San Diego, CA, USA) in the dark for 30 min at room temperature. After washing three times with 1 × PBS, HUMSCs were centrifuged at 2000 rpm for 5 min and resuspended in 1 × PBS for flow cytometry analysis. Cells were analyzed using a FACScan (BD FACSAria™; BD,San Jose, CA, USA) and data were analyzed with FACS software.

Phenotyping of resting and activated moDCs was performed by flow cytometry using anti-human CD1d-phycoerythrin (PE), CD103-FITC, HLA-DQ-FITC, PD-L1-PE (BD Biosciences, Franklin Lakes, NJ, USA), CD1a-allophycocyanin (APC), CD40-FITC (BioLegend, San Diego, CA, USA), CX₃CR1-PE, CD80-FITC, CD83-FITC, CD86-PE, DC-SIGN-FITC, CCR7-PE, CD14-PE (R&D Systems, Minneapolis, MN, USA), B7RP1 (ICOSL)-PE (EBiosciences, Santa Clara, CA, USA), and isotype-matched control antibodies. The ratio of regulatory T-lymphocytes was measured by flow cytometry using anti-human CD25-PE (BD Pharmingen), CD4-FITC (BioLegend), FoxP3-APC (R&D Systems), and anti-IL-10-AlexaFluor488 (BioLegend). The viability of moDCs was determined with 2 μg/ml 7-amino-actinomycin D (LKT Laboratories Inc., St. Paul, MN, USA) dye followed by a 24-h activation period with live bacteria or LPS. Fluorescence intensities were measured by FACSCalibur (BD Biosciences), and data were analyzed by the FlowJo software (Tree Star, Ashland, OR, USA).

Phenotyping of conditioned monocyte-derived DCs (moDCs) and macrophages was performed by flow cytometry, anti-human CD14-fluorescein isothiocyanate (FITC), CD209/DC-SIGN-phycoerythrin (PE), CD1a-FITC, CD80-FITC CD86-PE, PD-L1-PE, CTLA-4-PE, CD163-PE, CD206-PE (BioLegend), HLA-DQ-FITC (BD Biosciences) were used.