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. 2007;2(4):805-10.
doi: 10.1038/nprot.2007.111.

A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood

Dan G Duda  1 , Kenneth S Cohen, David T Scadden, Rakesh K Jain
Affiliations

A protocol for phenotypic detection and enumeration of circulating endothelial cells and circulating progenitor cells in human blood

Dan G Duda et al. Nat Protoc. 2007.

Abstract

Blood circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells (CPCs) represent two cell populations that are thought to play important roles in tissue vascularization. CECs and CPCs are currently studied as surrogate markers in patients for more than a dozen pathologies, including heart disease and cancer. However, data interpretation has often been difficult because of multiple definitions, methods and protocols used to evaluate and count these cells by different laboratories. Here, we propose a cytometry protocol for phenotypic identification and enumeration of CECs and CPCs in human blood using four surface markers: CD31, CD34, CD133 and CD45. This method allows further phenotypic analyses to explore the biology of these cells. In addition, it offers a platform for longitudinal studies of these cells in patients with different pathologies. The protocol is relatively simple, inexpensive and can be adapted for multiple flow cytometer types or software. The procedure should take 2-2.5 h, and is expected to detect 0.1-6.0% viable CECs and 0.01-0.20% CPCs within blood mononuclear cell population.

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Figures

Figure 1
Figure 1
Multicolor flow cytometric analyses of human mononuclear cells in whole blood samples. (a–d) Staining for CD31, CD34, CD133 and CD45 of a whole blood sample and gating on mononuclear cellular events on the forward-side scatter plot (in red in a) allow identification of two distinct populations of interest: CD31brightCD45−CD34+CD133− (green rectangles) and CD133+CD34brightCD31+CD45dim progenitor cells (blue, in yellow rectangles in b–d).
Figure 2
Figure 2
Multicolor flow cytometric analyses of human mononuclear cells separated from whole blood samples. (a–d) Staining for CD31, CD34, CD133 and CD45 of a whole blood sample and gating on mononuclear cellular events on the forward-side scatter plot (in red in a) allow identification of two distinct populations of interest: CD31brightCD45−CD34+CD133 − (green rectangles) and CD133+CD34brightCD31+CD45dim progenitor cells (blue, in yellow rectangles in b–d).
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