Quantifying Endothelial Progenitor Cells

Peripheral mononuclear cells (PMNCs) were fractionated using Ficoll density-gradient centrifugation. Isolated PMNCs were resuspended 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 nonadherent 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 [12 (link)]. Results are expressed as the mean number of colony-forming units (CFUs) per well.