This article is a US government work and, as such, is in the public domain in the United States of America.
Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells
Article first published online: 29 MAR 2013
Published 2013 Wiley-Periodicals, Inc.
Cytometry Part B: Clinical Cytometry
Volume 84, Issue 6, pages 379–389, November/December 2013
How to Cite
How to cite this article: Imaging flow cytometry for morphologic and phenotypic characterization of rare circulating endothelial cells. Cytometry Part B 2013; 84B: 379–389., , , , , .
- Issue published online: 25 OCT 2013
- Article first published online: 29 MAR 2013
- Manuscript Accepted: 2 MAR 2013
- Manuscript Revised: 4 FEB 2013
- Manuscript Received: 20 NOV 2012
- Division of Intramural Research
- National Heart Lung and Blood Institute, NIH, Bethesda, MD
- circulating endothelial cells;
- imaging cytometry;
- rare event cytometry
Endothelial cells in the peripheral circulation are rare events that require technically rigorous approaches for detection by flow cytometry. Visualization of these cells has been even more demanding, as this has historically required extensive enrichment and processing prior to attempting imaging. As a result, few, if any, examples exist on images of peripheral blood circulating endothelial cells (CECs) that include verification of the cell lineage both phenotypically and genomically. In this study, we have devised a method whereby CECs can be directly visualized after lysis of red blood cells and staining, without pre-enrichment or additional processing. Peripheral blood is stained with CD45, CD146, CD3, Hoechst, and DAPI to permit identification of CD146 positive, nonleukocyte, nucleated, and live cells that fit the description of CECs. These cells are imaged using the Amnis ImageStreamX, an imaging flow cytometer. Genomic verification of the endothelial nature of these cells is accomplished by using an aliquot of the same stained samples for sorting CECs using similar gating strategies. This proof of principle of direct imaging of CECs by imaging flow cytometry will permit studies to be conducted heretofore not possible, as the ImageStreamX has the capability of detecting additional fluorochromes other than those used to identify the CECs. Such potential investigations include antigen colocalization or capping, autophagy and apoptosis, morphologic changes in response to therapy, and others. Thus, this method will enable a broad range of novel studies to be conducted using CECs as surrogates of the endothelium. Published 2013 Wiley-Periodicals, Inc.†