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Cited by 357

The DOI matches its Crossref record; 77 of the 77 references it cites were found in a registry or an index.

PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles

Open access
Pužar Dominkuš, Pia, Stenovec, Matjaž, Sitar, Simona, Lasič, Eva, Zorec, Robert, Plemenitaš, Ana, Žagar, Ema, Kreft, Marko, Lenassi, Metka·2018·Biochimica et Biophysica Acta (BBA) - Biomembranes
DOI 10.1016/j.bbamem.2018.03.013
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Abstract

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis. We show for the first time that numerous PKH26 nanoparticles (nine out of ten PKH26-positive particles) are formed during ultracentrifugation-based exosome staining, which are almost indistinguishable from PKH26-labeled exosomes in terms of size, surface area, and fluorescence intensity. When PKH26-labeled exosomes were purified through sucrose, PKH26 nanoparticles were differentiated from PKH26-labeled exosomes based on their reduced size. However, PKH26 nanoparticles were only physically removed from PKH26-labeled exosomes when separated on a sucrose gradient, and at the expense of low PKH26-labeled exosome recovery. Overall, low PKH26-positive particle recovery is characteristic of filtration-based exosome staining. Importantly, PKH26 nanoparticles are internalized by primary astrocytes into similar subcellular compartments as PKH26-labeled exosomes. Altogether, PKH26 nanoparticles can result in false-positive signals for stained EVs that can compromise the interpretation of EV internalization. Thus, for use in EV uptake and functional studies, sucrose-gradient-based isolation should be the method of choice to obtain PKH26-labeled exosomes devoid of PKH26 nanoparticles.

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Crossref · 10.1016/j.bbamem.2018.03.013

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Metadata

Authors
Pužar Dominkuš, P., Stenovec, M., Sitar, S., Lasič, E., Zorec, R., Plemenitaš, A., Žagar, E., Kreft, M., & Lenassi, M.
Venue
Biochimica et Biophysica Acta (BBA) - Biomembranes
Publisher
Elsevier BV
Published
2018-06
Volume
1860
Issue
6
Pages
1350-1361
ISSN
0005-2736 (ISSN-L)
DOI
10.1016/j.bbamem.2018.03.013
OpenAlex
W2791527635
Cited by
357
References
77
FWCI
8.63
Type
journal article
Language
en
Open access
bronze

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The paper
Pužar Dominkuš, P., Stenovec, M., Sitar, S., Lasič, E., Zorec, R., Plemenitaš, A., Žagar, E., Kreft, M., & Lenassi, M. (2018). PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles. Biochimica et Biophysica Acta (BBA) - Biomembranes. https://doi.org/10.1016/j.bbamem.2018.03.013

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Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.