Comprehensive phenotyping of endothelial cells using flow cytometry 2: Human

Abstract

In vascular research, clinical samples and samples from animal models are often used together to foster translation of preclinical findings to humans. General concepts of endothelia and murine-specific endothelial phenotypes were discussed in part 1 of this two part series. Here, in part 2, we present a comprehensive overview of human-specific endothelial phenotypes. Pan-endothelial cell markers, organ specific endothelial antigens, and flow cytometric immunophenotyping of blood-borne endothelial cells are reviewed.

Keywords: endothelial cell; endothelial cell subset; flow cytometry; human; immunophenotyping; phenotyping.

Figure 1.. Example Gating Strategy for Identification of Human Circulating Endothelial Cells

Gating of CECs in human peripheral blood. Whole blood processed using RBC-lysis/WBC fixation protocol, followed by cryopreservation . Thawed cells were used for flow cytometry. Cell labeling is described in detail in the supplemental file. The gating is illustrated here. Artifacts are excluded by first gating based on time to eliminate fluidic disturbances (A), then using forward scatter height and forward scatter area, aggregates are excluded (B). Cell debris is excluded (C) before gating based on DAPI to select cells in G₀/G₁ (D). Hematopoietic cells are excluded by selecting CD45− cells (E) based on the fluorescence minus one (FMO) control containing all markers except CD45 (F). The pan-endothelial marker CD31 is used to identify all circulating endothelial cells and cells double positive for CD31 and CD34 are selected as a specific subset (G). The gating boundaries in plot G were determined using the FMO controls for CD34 (H) and CD31 (I). 0.86% of all circulating cells were CD31+ CECs and 0.03% were the CD31+ CD34+ subset. In this example, fixed cells were used. A cell membrane permeable DNA-binding dye should be utilized when working with unfixed cells. A viability dye is also highly recommended if a dump channel, like CD45 in this example, is not used.