Anti cd105 fitc

We used trypsin (0.05%) and EDTA (0.02%) to remove the MSCs and differentiated endothelial cells from the culture flasks. The cells were counted with a Neubauer slide and cell viability determined with trypan blue staining. The cell suspension (10⁶ cells/ml) was prepared in 50 μl PBS and incubated with either fluorescein isothiocyanate (FITC) or PE-conjugated antibodies, in the dark for 45 minutes at 4˚C. Immud nophenotyping of the MSCs was carried out using anti-CD44-PE, anti-CD166-PE, anti-CD105-FITC, and anti-CD34-FITC antibodies (eBioscience, USA). In order to immunophenotype the endothelial cells before and after differentiation, we incubated the MSCs and differentiated endothelial cells with anti-FLT1FITC, anti-VE-cadherin-FITC, anti-VCAM1-FITC, anti-Tie2-FITC, anti-vWF-FITC, anti-CD31-FITC, and anti-VEGFR2-FITC antibodies (eBioscience, USA). Next, the cells were washed twice with PBS that contained 2% bovine serum albumin (BSA, Gibco Invitrogen, USA) and fixed with 1% paraformaldehyde solution in PBS. Mouse isotype antibodies served as the negative control. Analysis was performed using a flow cytometer (Partech, Germany).