Lipid-based strategies used to identify extracellular vesicles in flow cytometry can be confounded by lipoproteins: Evaluations of annexin V, lactadherin, and detergent lysis

Jaco Botha*, Aase Handberg, Jens B. Simonsen*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

148 Downloads (Pure)

Abstract

Flow cytometry (FCM) is a popular method used in characterisation of extracellular vesicles (EVs). Circulating EVs are often identified by FCM by exploiting the lipid nature of EVs by staining with Annexin V (Anx5) or lactadherin against the membrane phospholipid phosphatidylserine (PS) and evaluating the specificity of the labels by detergent lysis of EVs. Here, we investigate whether PS labelling and detergent lysis approaches are confounded by lipoproteins, another family of lipid-based nanoparticles found in blood, in both frozen and fresh blood plasma. We demonstrated that Anx5 and lactadherin in addition to EVs stained ApoB-containing lipoproteins, identified by the use of fluorophore-labelled polyclonal ApoB-antibody, and that Anx5 had a significantly larger tendency for labelling lipoprotein-bound PS than lactadherin. Furthermore, detergent lysis resulted in a decrease in both EV and lipoprotein events and especially lipoproteins positive for either Anx5 or lactadherin. Taken together, our findings pose concerns to the use of lipid-based strategies in identifying EVs by FCM and support the use of transmembrane proteins such as tetraspannins to distinguish EVs from lipoproteins.
Original languageEnglish
Article numbere12200
JournalJournal of Extracellular Vesicles
Volume11
Issue number4
Number of pages15
ISSN2001-3078
DOIs
Publication statusPublished - 2022

Keywords

  • Annexin V
  • Chylomicrons
  • Detergent lysis
  • Exosomes
  • Extracellular vesicles
  • Flow cytometry
  • Lactadherin
  • Lipids
  • Lipoproteins
  • Phosphatidylserine
  • Triton X-100
  • VLDL

Fingerprint

Dive into the research topics of 'Lipid-based strategies used to identify extracellular vesicles in flow cytometry can be confounded by lipoproteins: Evaluations of annexin V, lactadherin, and detergent lysis'. Together they form a unique fingerprint.

Cite this