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To characterize monocyte subsets, freshly purified PBMCs were analyzed by six-color flow cytometry on FACS LSRII apparatus. The gating strategy was based on a previous report [15 (link)]. Monocytes were subdivided into three major subsets: classical CD14⁺⁺CD16⁻, intermediate CD14⁺⁺CD16⁺ and non-classical CD14⁺CD16⁺⁺ monocytes. The following anti-human mAbs were used: CD45-Amcyan (BD Biosciences), HLA-DR-PerCP (BD Biosciences), CD19-ECD (Beckman Coulter), CD14-QDot655 (Invitrogen), CD16-APC-H7 (Beckman Coulter, Villepinte, France). The Live/Dead blue Dye (Invitrogen) was used to exclude dead cells.
Samples of the purified monocytes used to generate MD-DCs and of the resulting MD-DCs were routinely stained with the following anti-human mAbs: CD14-FITC, CD11c-APC, CD40-PE, HLA-I-FITC, HLA-DR-PerCP, CD80-PE, CD83-APC and CD86-FITC (all from BD Bioscience) and analyzed by flow cytometry on FACS canto II apparatus (BD Biosciences).

Immunophenotypic characterization of MSCs was performed according to the methodology described by Montesinos et al. [16 (link)]. Monoclonal antibodies against surface markers characteristic of MSCs were used: CD105-PE, CD90-APC, CD73-PE, HLA-I-FITC, HLA-II-PE, and CD45-APC (BD Biosciences, San Diego, CA); CD13-PE and CD14-PE (Caltag, Buckingham, United Kingdom); and CD31-FITC and CD34-APC (Invitrogen, Carlsbad, CA). A total of 1‐1.5 × 10⁶ MSCs were resuspended in 100 mL of phosphate-buffered saline with 3% FBS and 1 mM EDTA (cytometry buffer) and incubated for 20–30 min with the appropriate antibodies. Next, the cells were washed with 1 mL of buffer and fixed with FACS Lysing Solution (BD Biosciences). The samples were analyzed on a Coulter Epics Altra Flow Cytometer (Beckman Coulter, Brea, CA), and at least 10,000 events were collected. The percentages of positive cells and mean fluorescence intensity (MFI) were obtained. The data were analyzed with FlowJo 7.6.1 software.