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

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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, the live cell fluorescence lifetime imaging.

Original languageEnglish
Pages (from-to)226-240
Number of pages15
JournalNanoscale Advances
Issue number1
Early online date23 Nov 2021
Publication statusPublished - 7 Jan 2022
Publication typeA1 Journal article-refereed

Publication forum classification

  • Publication forum level 1

ASJC Scopus subject areas

  • Bioengineering
  • Chemistry(all)
  • Atomic and Molecular Physics, and Optics
  • Materials Science(all)
  • Engineering(all)


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