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. 2017 Dec 19;7:17880. doi: 10.1038/s41598-017-18156-6

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Flow cytometry gating strategy to quantify endothelial progenitor cells (EPCs). Initially, debris, red blood cells and platelets were removed from the analysis based on their forward scatter (FCS) vs. side scatter (SSC) properties (R0 plot 1) and then a gate was set on a CD45 vs. dot plot to contain all CD45⁺ events (R1 plot 2). Next, gate R1 events were displayed on a CD34 vs. SSC dot plot (plot 3) and a second gate (R2) was defined in a sequential strategy to include CD34⁺ events. CD34⁺ cells with low SSC and low CD45 fluorescence (SSC^low/CD45^dim cells) were then gated (R3) (plot 4). The low SSC properties and expression of CD45 were confirmed against total events. Finally, EPCs were identified by a gate (R4) set on a CD34 vs. CD309 (VEGFR2/KDR) dot plot and were defined as CD45^dim/CD309⁺/CD34⁺.