Pe conjugated mouse anti human cd105

For cell surface markers analyses, cells were treated with 0.5% (w/v) bovine serum albumin and double-stained using monoclonal antibodies, including phycoerythrin (PE)-conjugated mouse anti-human CD34 (BD Biosciences, Le-Pont-de-Claix, France) with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD90 (Beckman Coulter, Villepinte, France), PE-conjugated mouse anti-human CD166 (Beckman Coulter, Villepinte, France) with FITC-conjugated mouse anti-human CD45 (Dako, Glostrup, Denmark), PE-conjugated mouse anti-human CD105 (Beckman Coulter, Villepinte, France) with FITC-conjugated mouse anti-human CD44 (Beckman Coulter, Villepinte, France), and PE-conjugated mouse anti-human CD73 (BD Biosciences, Le-Pont-de-Claix, France) with FITC-conjugated mouse anti-human leukocyte antigen-antigen D related (HLA-DR) (Beckman Coulter, Villepinte, France). Antibodies were used at a dilution of 1:10 (PE CD34, FITC CD90, PE CD166, FITC CD44, PE CD105, FITC HLA-DR and PE CD73) or 1:20 (FITC CD45). Appropriate isotype-matched control antibodies named FITC or PE mouse IgG1 (Dako, Glostrup, Denmark) were used in each analysis. Cells were then examined by flow cytometry using a BD LSR II flow cytometer (Becton Dickinson Biosciences, San Jose, California). Fluorescence intensity and percentage of antigen positive cells were determined for each surface marker.

Flow cytometry was used to characterize the surface markers of the cultured EC-MSCs. Cells from P3 were dissociated from the growth flask using Hanks'-Based, Enzyme Free, Cell Dissociation Buffer (Gibco), and resuspended in DMEM (Lonza) containing 10 % FBS (Gibco). Samples were counted, centrifuged, and resuspended in PBS containing 10 % FBS. The cells were transferred to Eppendorf tubes at 5 × 10⁵ cells per 1 mL, washed twice with PBS containing 10 % FBS, and incubated for 1 h at room temperature in the dark with the following antibodies: PE-conjugated mouse anti-human CD73 (Clone AD2, BD Pharmingen), PE-conjugated mouse anti-human CD90 (Clone 5E10, BD Pharmingen, Erembodegem, Belgium), PE-conjugated mouse anti-human CD105 (Clone 1G2, Beckman Coulter, Marseille, France), and FITC-conjugated rat anti-mouse CD45 (Clone 30-F11, eBioscience, Halle-Zoersel, Belgium). The samples were then washed twice with PBS, stained with 1 μL of Fixable Viability Dye eFluor® 450 per 1 mL of cells, vortexed, incubated for 30 min at 4 °C in the dark, and washed with PBS before analysing with flow cytometry. Unlabelled cells were used as the negative control for detection of autofluorescence.