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Mouse ES cells (HM1) were differentiated into embryoid bodies (EBs). At day 5 EBs were dissociated with the Embryoid Body Dissociation Kit (# 130-096-348) and CD309 expressing cells were isolated using the CD309 (Flk-1) MicroBead Kit, an LS Column, and a MidiMACS™ Separator. After fluorescent labeling with Labeling Check Reagent‑APC (# 130-095-237) cells were analyzed by flow cytometry using the MACSQuant®
Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.
Improved endothelial differentiation of isolated CD309 (Flk-1) + cells:
Unseparated or CD309-enriched cardiovascular progenitor cells (derived as in fig. 1) were subjected to endothelial differentiation in the presence of 50 ng/mL VEGF. Differentiation efficiency was evaluated by immunofluorescence using CD31-FITC (green) and DAPI counterstain (blue).
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