Improved characterization of EV preparations based on protein to lipid ratio and lipid properties

Xabier Osteikoetxea, Andrea Balogh, Katalin Szabó-Taylor, Andrea Németh, Tamás Géza Szabó, Krisztina Pálóczi, Barbara Sódar, A. Kittel, Bence György, E. Pállinger, J. Matkó, E. Búzás

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies.

Original languageEnglish
Article numbere0121184
JournalPLoS One
Volume10
Issue number3
DOIs
Publication statusPublished - Mar 23 2015

Fingerprint

Lipids
lipids
Proteins
proteins
lipid bilayers
cholesterol
lipid composition
Assays
Lipid bilayers
assays
Cholesterol
Lipid Bilayers
exosomes
gangliosides
cholera toxin
Extracellular Vesicles
blood plasma
Exosomes
G(M1) Ganglioside
quality control

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Osteikoetxea, X., Balogh, A., Szabó-Taylor, K., Németh, A., Szabó, T. G., Pálóczi, K., ... Búzás, E. (2015). Improved characterization of EV preparations based on protein to lipid ratio and lipid properties. PLoS One, 10(3), [e0121184]. https://doi.org/10.1371/journal.pone.0121184

Improved characterization of EV preparations based on protein to lipid ratio and lipid properties. / Osteikoetxea, Xabier; Balogh, Andrea; Szabó-Taylor, Katalin; Németh, Andrea; Szabó, Tamás Géza; Pálóczi, Krisztina; Sódar, Barbara; Kittel, A.; György, Bence; Pállinger, E.; Matkó, J.; Búzás, E.

In: PLoS One, Vol. 10, No. 3, e0121184, 23.03.2015.

Research output: Contribution to journalArticle

Osteikoetxea, X, Balogh, A, Szabó-Taylor, K, Németh, A, Szabó, TG, Pálóczi, K, Sódar, B, Kittel, A, György, B, Pállinger, E, Matkó, J & Búzás, E 2015, 'Improved characterization of EV preparations based on protein to lipid ratio and lipid properties', PLoS One, vol. 10, no. 3, e0121184. https://doi.org/10.1371/journal.pone.0121184
Osteikoetxea X, Balogh A, Szabó-Taylor K, Németh A, Szabó TG, Pálóczi K et al. Improved characterization of EV preparations based on protein to lipid ratio and lipid properties. PLoS One. 2015 Mar 23;10(3). e0121184. https://doi.org/10.1371/journal.pone.0121184
Osteikoetxea, Xabier ; Balogh, Andrea ; Szabó-Taylor, Katalin ; Németh, Andrea ; Szabó, Tamás Géza ; Pálóczi, Krisztina ; Sódar, Barbara ; Kittel, A. ; György, Bence ; Pállinger, E. ; Matkó, J. ; Búzás, E. / Improved characterization of EV preparations based on protein to lipid ratio and lipid properties. In: PLoS One. 2015 ; Vol. 10, No. 3.
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