Enumeration of Circulating Endothelial Cells and Progenitors

100 μl of whole blood was incubated with 5 μl of V500 CD45 (BD Biosciences, Oxford, UK), 20 μl of PerCP-Cy5.5 CD34 (BD Biosciences, Oxford, UK), 5 μl of PE VEGFR-2+ (R&D Systems, Roche Diagnostics, West Sussex, UK), 5 μl APC CD133 (BD Biosciences, Oxford, UK), 10 μl FITC CD144 (BD Biosciences, Oxford, UK) for 30 min. Subsequently, 2 ml of pharmlyse (BD Biosciences, Oxford, UK) was used to lyse the red cells. The sample was then analysed by flow cytometry on BD FACS Canto™ II system and samples were run to approximately 500,000 total events. Analysis was carried out using BD FACSDiva™ software. On average 440,000 events were counted. CEPCs events were defined using the most recent definition of CD45^dimCD34⁺ VEGFR2 (KDR)⁺, as recommended by Van Craenenbroeck et al. [21 (link)]. Intra-assay variation (CD45^dimCD34⁺VEGFR-2⁺) was less than 8 %. The results were expressed as % leukocytes [22 (link), 21 (link)]. CECs events were defined as CD45^dimCD133^-CD34⁺CD144⁺ [23 (link), 24 (link)].