Pe conjugated mouse anti human cd90

Flow cytometry was used to analyze the expression of cell surface markers in RFP- and Oct4/Sox2-ATMSCs. By passage 5, a homogenous population of rapidly dividing cells with fibroblastoid morphology was obtained. ATMSCs were fixed with 70% ethanol at 4°C and stained for 30 min on ice with primary antibodies that recognized various surface molecules. Phycoerythrin-conjugated (PE) mouse anti-human CD29 (BD Bioscience, San Jose, CA), fluorescein isothiocyanate-conjugated (FITC) mouse anti-human CD31 (BD Bioscience), PE-conjugated mouse antihuman CD34 (BD Bioscience), FITC-conjugated mouse anti-human CD44 (BD Bioscience), FITC-conjugated mouse anti-human CD45 (BD Bioscience), PE-conjugated mouse anti-human CD73 (BD Biosciences Pharmingen, San Diego, CA), PE-conjugated mouse anti human CD90 (BD Biosciences), and PE-conjugated antihuman CD105/endoglin (R&D System, Minneapolis, MN) were used for the detection of cell surface antigens. The immunophenotype of MSCs was analyzed with the FACSCalibur flow cytometer (BD Biosciences, Bedford, MA) using the CELLQuest software (BD Biosciences).

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.