Identification of Endothelial Progenitor Cells

For identification of EPCs derived from peripheral blood, flow cytometry was applied for the assessment of EPC surface markers, including sets of KDR⁺/CD34⁺/CD45⁻, CD133⁺/CD34⁺/CD45⁻, and CD34⁺, as described in our recent reports [6 (link), 8 (link), 11 (link)]. Briefly, mononuclear cells were isolated by density-gradient centrifugation of Ficoll-Paque Plus™ (GE Healthcare Biosciences, Uppsala, Sweden) and further incubated with fluorescent dye-conjugated mononuclear antibodies. After incubation, the mononuclear cells were fixed in 1% of paraformaldehyde for flow cytometry analysis by using a fluorescence-activated cell sorter (FACSCalibur system; Beckman Coulter, CA, USA). The assays for circulating EPCs in each sample were performed in duplicate, and the mean levels were reported.