Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles

Kaisa Rautaniemi, Jacopo Zini, Emilia Löfman, Heikki Saari, Iida Haapalehto, Johanna Laukka, Sami Vesamäki, Alexander Efimov, Marjo Yliperttula, Timo Laaksonen, Elina Vuorimaa-Laukkanen, Ekaterina S. Lisitsyna

Tutkimustuotos: ArtikkeliScientificvertaisarvioitu

20 Lataukset (Pure)

Abstrakti

Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameterErp(relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application,e.g.in the live cell fluorescence lifetime imaging.

AlkuperäiskieliEnglanti
Sivut226-240
Sivumäärä15
JulkaisuNanoscale Advances
Vuosikerta4
Numero1
Varhainen verkossa julkaisun päivämäärä23 marrask. 2021
DOI - pysyväislinkit
TilaJulkaistu - 7 tammik. 2022
OKM-julkaisutyyppiA1 Alkuperäisartikkeli tieteellisessä aikakauslehdessä

Julkaisufoorumi-taso

  • Jufo-taso 1

!!ASJC Scopus subject areas

  • Bioengineering
  • Yleinen kemia
  • Atomic and Molecular Physics, and Optics
  • Yleinen materiaalitiede
  • Yleinen tekniikka

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