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Filename: controllers/Detail.php
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Background: Extracellular vesicles (EVs) in biofluids are potential biomarkers of disease. To explore the clinical relevance of EVs, a specific generic EV marker would be useful, one that does not require antibodies and binds to all EVs. Here we evaluated 5 commonly used generic markers for flow cytometry.
Methods: Flow cytometry (A60-Micro, Apogee) was used to evaluate the ability of the generic EV markers calcein acetoxymethyl ester, calcein acetoxymethyl ester violet, carboxyfluorescein succinimidyl ester (CFSE), 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium (di-8-ANEPPS), and lactadherin to stain EVs from MCF7 human breast adenocarcinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM)] or platelet EVs from human plasma [integrin β3 positive (CD61)]. Side scatter triggering was applied as a reference, and the influence of non-EV components (proteins and lipoproteins) was evaluated.
Results: Di-8-ANEPPS, lactadherin, and side scatter detected 100% of EpCAM MCF7 EVs. Lactadherin and side scatter detected 33% and 61% of CD61 EVs, respectively. Di-8-ANEPPS detected platelet EVs only if soluble protein was first removed. Because all generic markers stained proteins, at best 33% of platelet EVs in plasma were detected. The calcein markers and CFSE were either insensitive to EVs in both samples or associated with swarm detection.
Conclusions: None of the generic markers detected all and only EVs in plasma. Side scatter triggering detected the highest concentration of plasma EVs on our A60-Micro, followed by lactadherin. The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used.
Download full-text PDF |
Source |
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http://dx.doi.org/10.1373/clinchem.2017.278978 | DOI Listing |
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