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

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

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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
Volume64
Issue number4
DOIs
Publication statusPublished - Apr 1 2018

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Flow cytometry
Platelets
Flow Cytometry
Cell Adhesion Molecules
Plasmas
Esters
Plasma (human)
Proteins
Biomarkers
Conditioned Culture Medium
Integrins
Lipoproteins
Coloring Agents
Cells
Antibodies
Blood Platelets
1-(3-sulfonatopropyl)-4-(beta-(2-(di-n-octylamino)-6-naphthyl)vinyl)pyridinium betaine
Extracellular Vesicles
calcein AM
6-carboxyfluorescein

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., ... Coumans, F. A. W. (2018). Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry. Clinical Chemistry, 64(4), 680-689. https://doi.org/10.1373/clinchem.2017.278978

Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry. / De Rond, Leonie; Van Der Pol, Edwin; Hau, Chi M.; Varga, Z.; Sturk, Auguste; Van Leeuwen, Ton G.; Nieuwland, Rienk; Coumans, Frank A.W.

In: Clinical Chemistry, Vol. 64, No. 4, 01.04.2018, p. 680-689.

Research output: Contribution to journalArticle

De Rond, L, Van Der Pol, E, Hau, CM, Varga, Z, Sturk, A, Van Leeuwen, TG, Nieuwland, R & Coumans, FAW 2018, 'Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry', Clinical Chemistry, vol. 64, no. 4, pp. 680-689. https://doi.org/10.1373/clinchem.2017.278978
De Rond, Leonie ; Van Der Pol, Edwin ; Hau, Chi M. ; Varga, Z. ; Sturk, Auguste ; Van Leeuwen, Ton G. ; Nieuwland, Rienk ; Coumans, Frank A.W. / Comparison of generic fluorescent markers for detection of extracellular vesicles by flow cytometry. In: Clinical Chemistry. 2018 ; Vol. 64, No. 4. pp. 680-689.
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abstract = "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.",
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AU - De Rond, Leonie

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AU - Sturk, Auguste

AU - Van Leeuwen, Ton G.

AU - Nieuwland, Rienk

AU - Coumans, Frank A.W.

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N2 - 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.

AB - 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.

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