Pitfalls associated with lipophilic fluorophore staining of extracellular vesicles for uptake studies

Post-staining of extracellular vesicles (EVs) with lipid-anchored fluorophores (LAFs) such as PKH67 is a widely used strategy for studying EVs but it is associated with several pitfalls. The pitfalls discussed in this commentary are related to LAF labelling of non-EV species due to (1) lipoprotein contamination in EV samples, (2) desorption of the LAF reporters from vesicles into proteins and lipoproteins in blood and serum, and (3) the capability of the amphiphilic LAF compounds to form EV-like particles. Awareness of these challenges and developing solutions to overcome these are important to ensure that we make relevant interpretations when using LAFs to track EVs.

Schematic illustration of the proposed paths of the lipid-anchored fluorophore (LAF) used for labelling and tracking EVs that are discussed in this commentary

(a) LAF labelling of non-EV serum components, that are often present in EV isolates, in addition to EV labelling. (b) LAF labelling of pure EVs. (c) Dissociation of LAFs from EVs into serum components. (d) Formation of LAF particles that exhibit low fluorescence due to self-quenching.