Peripheral mononuclear cells (PMNCs) were fractionated using Ficoll density-gradient centrifugation. Isolated PMNCs were re-suspended in CFU-Hill Medium (Stem Cell Technologies, Vancouver, Canada) and plated on 6-well plates, coated with human fibronectin at a concentration of 5×106 cells per well. After 48 hours, the non-adherent cells were collected and replated onto fibronectin-coated 24-well plates. EPC colonies were counted using an inverted microscope 7 days after plating. An EPC colony was defined as a cluster of at least 100 flat cells surrounding a cluster of rounded cells, as previously described [11 (link)] . A central cluster alone without associated emerging cells was not counted as a colony. Results are expressed as the mean number of colony-forming units (CFUs) per well.
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Isolation and Quantification of Endothelial Progenitor Cells
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Corresponding Organization : NewYork–Presbyterian Brooklyn Methodist Hospital
Other organizations : Marshall University, New York Medical College
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Variable analysis
independent variables
- Ficoll density-gradient centrifugation to fractionate peripheral mononuclear cells (PMNCs)
- Number of endothelial progenitor cell (EPC) colonies counted using an inverted microscope 7 days after plating
- Plating of isolated PMNCs on 6-well plates coated with human fibronectin at a concentration of 5×10^6 cells per well
- Collection of non-adherent cells after 48 hours and re-plating onto fibronectin-coated 24-well plates
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