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Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential.

ABSTRACT: Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34(+)CD45(dim) cells representing functional circulating hematopoietic stem and progenitor cells (CHSPCs) that can be separated on the basis of AC133 expression and report that the AC133(+) subset of the CHSPCs enhances the growth of tumor blood vessels in vivo in immunodeficient mice. In addition, the ratio of AC133(+) proangiogenic CHSPCs to AC133(-) nonangiogenic CHSPCs unambiguously correlates with the severity of the clinical state of patients with peripheral arterial disease. In sum, a PFC protocol validated via in vitro and in vivo analyses, can be used to interrogate the roles of human hematopoietic elements in the growth and maintenance of the vasculature.

SUBMITTER: Estes ML

PROVIDER: S-EPMC2931367 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential.

Estes Myka L ML Mund Julie A JA Mead Laura E LE Prater Daniel N DN Cai Shanbao S Wang Haiyan H Pollok Karen E KE Murphy Michael P MP An Caroline S T CS Srour Edward F EF Ingram David A DA Case Jamie J

Cytometry. Part A : the journal of the International Society for Analytical Cytology 20100901 9

Defining whether human circulating proangiogenic cells represent a subset of the hematopoietic system and express CD45 or are hematopoietic derivatives that do not express CD45 (and are called endothelial progenitor cells) remains controversial. We have previously developed a polychromatic flow cytometry (PFC) protocol to isolate subsets of hematopoietic cells and we now identify the circulating pool of CD34(+)CD45(dim) cells representing functional circulating hematopoietic stem and progenitor ...[more]

PMID: 20803735

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Project description:BackgroundNatural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/- and CD56DimCD16+ NK cells. Intracellular calcium (Ca2+) is fundamental to regulate a number of intracellular signalling pathways and functions in NK cells, which are essential in mediating their natural cytotoxic function. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable non-selective cation channel that possesses a critical role in calcium-dependent cell signalling to maintain cellular homeostasis. TRPM2 and CD38 protein surface expression has yet to be determined on NK cells using flow cytometry. Characterisation of TRPM2 has been previously identified by in vivo models, primarily using methods such as genetic remodification, immunohistochemistry and whole cell electrophysiology. The aim of this study was to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously applied using flow cytometry.ResultsAt 2 h/1 h, TRPM2 (Fig. 2 A, B, p < 0.05) and TRPM2/CD38 (Fig. 3A, B, p < 0.05) surface expression significantly increased between 1:300 and 1:50 at 2 h/1 h. TRPM2/CD38 surface expression furthermore increased between 1:100 and 1:50 at 2 h/1 h (Fig. 3A, p < 0.05). Interestingly, TRPM2/CD38 surface expression significantly decreased from 1:50 to 1:5 on CD56BrightCD16Dim/- NK cells. These consistent findings highlight that 1:50 is the optimal antibody dilution and threshold to measure TRPM2 and TRPM2/CD38 surface expression on NK subsets. 2 h/1 h was determined as the optimal incubation period to ensure a sufficient timeframe for maximal antibody binding and surface expression.ConclusionFor the first time, we describe an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cells in healthy participants. Finally, using an antibody that has not been previously applied in flow cytometry, we determined an antibody concentration and incubation time that is robust, rapid and sensitive for the application of flow cytometry.

Dataset Information

Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential.

Publications

Application of polychromatic flow cytometry to identify novel subsets of circulating cells with angiogenic potential.

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