MSCs between passages 2 to 5 were characterized as CD14−CD45−CD44+CD90+ and differentiated as previously described by our group (Pankajakshan et al., 2012). The same pre-defined MSC type populations (>95% pure) were used for all subsequent FACS experiments. The same gating strategy was employed across all the FACS experiments. Briefly, cells (1 × 106 /ml) were washed with PBS containing 4% FBS and incubated with primary antibodies conjugated to fluorophores for 30 min at 4°C in the dark. The dilution and concentration of antibodies followed the specifications suggested by the manufacturer. The cells were further washed 3 times in PBS and re-suspended in 750 μl FACS-FIX. FACS analysis was acquired on a BD FACSAria I/II System (BD Biosciences, San Jose, CA, USA). As opposed to naïve MSCs, differentiated MSCs were highly positive for the following fluorophore-conjugated antibodies: vWF-PE (R&D Systems, Minneapolis, MN), VE-cadherin-FITC and PECAM-1-AP (eBioscience, San Diego, CA).
Ve cadherin fitc
Manufactured by Thermo Fisher Scientific
VE-cadherin-FITC is a fluorescently labeled antibody that specifically targets the VE-cadherin protein, which is a marker for vascular endothelial cells. This product can be used for flow cytometry and immunofluorescence applications to identify and quantify endothelial cells in various samples.
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2 protocols using ve cadherin fitc
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Characterization of Differentiated MSCs
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Characterization of Mesenchymal Stem Cell Differentiation
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Flow cytometry was performed using standard methods and carried out on a BD FACSAria I/II System (BD Biosciences, San Jose, CA). First, cells (~1 × 106/mL) were washed with PBS containing 4% FBS and incubated with primary antibodies conjugated to fluorophore (FITC) for 30 min at 4°C in the dark. The antibody concentrations followed the specifications of the manufacturer. After 3 washes in PBS, cells were resuspended in FACS-FIX. MSCs were characterized as CD14−CD45−CD44+CD73+CD90+. At the end of the 10-day differentiation protocol, MSCs were analyzed for the EC markers PECAM-1-APC (ebiosciences, San Diego, CA), VE-cadherin-FITC (ebiosciences, San Diego, CA), and vWF-PE (R&D Systems, Minneapolis, MN).
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