Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry

Leonie De Rond, Edwin Van Der Pol, Chi M. Hau, Zoltan Varga, Auguste Sturk, Ton G. Van Leeuwen, Rienk Nieuwland, Frank A.W. Coumans

Research output: Contribution to journalArticle

20 Citations (Scopus)


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 adenocar-cinoma cell line-conditioned culture medium [epithelial cell adhesion molecule positive (EpCAM)] or platelet EVs from human plasma [integrin 3 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 b lactadherin The choice between scatter or lactadherin primarily depends on the analytical sensitivity of the flow cytometer used.

Original languageEnglish
Pages (from-to)680-689
Number of pages10
JournalClinical chemistry
Issue number4
Publication statusPublished - Apr 2018


ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

De Rond, L., Van Der Pol, E., Hau, C. M., Varga, Z., Sturk, A., Van Leeuwen, T. G., Nieuwland, R., & Coumans, F. A. W. (2018). Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry. Clinical chemistry, 64(4), 680-689.