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An optimized flow cytometry protocol for analysis of angiogenic monocytes and endothelial progenitor cells in peripheral blood
Author(s) -
Hristov Mihail,
Schmitz Susanne,
Schuhmann Christoph,
Leyendecker Thorsten,
von Hundelshausen Philipp,
Krötz Florian,
Sohn HaeYoung,
Nauwelaers Frans A.,
Weber Christian
Publication year - 2009
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20772
Subject(s) - flow cytometry , cd34 , progenitor cell , angiogenesis , cd14 , cd16 , biology , angiopoietin receptor , immunology , endothelial progenitor cell , peripheral blood mononuclear cell , pathology , microbiology and biotechnology , medicine , cancer research , antigen , stem cell , cd8 , cd3 , biochemistry , in vitro
Circulating adult CD34 + VEGFR2 + endothelial progenitor cells (EPCs) have been shown to differentiate into endothelial cells, thus contributing to vascular homeostasis. Furthermore, a subset of circulating CD14 + monocytes coexpresses CD16 together with the angiopoietin receptor Tie2 and has been functionally implicated in tumor angiogenesis. However, clinically applicable protocols for flow cytometric quantification of EPCs and Tie2 + monocytes in peripheral blood and a consensus on reference values remain elusive. The number of Tie2 + CD14 + CD16 mid angiogenic monocytes and CD34 + VEGFR2 + CD45 low/− EPCs was assessed in the peripheral venous blood of patients with stable coronary artery disease by three‐color flow cytometry using specific monoclonal antibodies conjugated to PerCP, PE, PE‐Cy7, APC, and APC‐Cy7. Scatter multigating with exclusion of dead cells was performed to dissect complex mononuclear cell populations. This analysis was further refined by matching bright fluorochromes (PE‐Cy7, PE, APC) with dimly expressed markers (CD34, VEGFR2, Tie2), by automatic compensation for minimizing fluorescence spillover and by using fluorescence‐minus‐one (FMO) controls to determine positive/negative boundaries. Presuming a Gaussian distribution, we obtained average values (mean ± SD) of 1.45 ± 1.29% for Tie2 + CD14 + CD16 mid monocytes ( n = 11, range: 0.12–3.64%) and 0.019 ± 0.013% for CD34 + VEGFR2 + CD45 low/− EPCs ( n = 17, range: 0.003–0.042%). The intra‐ and inter‐assay variability was 1.6% and 4.5%, respectively. We have optimized a fast and sensitive assay for the flow cytometric quantification of circulating angiogenic monocytes and EPCs in cardiovascular medicine. This protocol may represent a basis for standardized analysis and monitoring of these cell subsets to define their normal range and prognostic/diagnostic value in clinical use. © 2009 International Society for Advancement of Cytometry