Quantification of Circulating Endothelial Progenitor Cells

Circulating EPCs were quantified using the method devised by Duda et al. [42 (link)], with a few modifications described in our previous study [43 (link)]. Flow cytometry was used for whole blood analysis without enrichment procedures to avoid manipulation artifacts. The EPCs were characterized as CD31⁺/vascular endothelial growth factor-2 (VEGFR-2)⁺/CD45^dim/CD133⁺. The chromophores conjugated with specific antibodies used in this study included CD31-FITC (BD Pharmingen, San Diego, CA), VEGFR2-PE (BD Pharmingen), CD45-PerCP (BD Pharmingen), and CD133-PE (Miltenyi Biotec, Auburn, CA). During the analysis of flow cytometry data, the mononuclear cell population was gated to avoid red blood cell, platelet, cell debris, and neutrophil contamination; 100,000 events in the gated population were collected using a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). The acquisition data were collected and analyzed using CellQuest Software (BD Biosciences). Representative data for identification and quantification of EPCs by flow cytometric analysis is shown in Figure 4.