Peripheral blood-derived endothelial progenitor cells (Huazhong University of Science and Technology)

Mononuclear cells (MNCs) were separated from the blood sample using Ficoll density gradient centrifugation. After isolation, MNCs were washed twice with Medium 199 (M199) and re-suspended in growth medium supplemented with M199 and  fetal calf serum (FBS, 20%), VEGF (10 ng/ml), FGF-2(2 ng/ml), penicillin (100 U/milliliter), and streptomycin (100 μg/milliliter). MNCs were then plated on human fibronectin-coated plates. In order to avoid contamination with mature endothelial cells, the nonadherent cells were collected 48 h thereafter and replated onto fibronectin-coated 24-well plates for a final assessment of the number of colonies. Growth medium was changed every 3 days.