Negligible incorporation of lipophilic dyes into bona fide small extracellular vesicles

Lipophilic dyes are widely used to track extracellular vesicles (EVs), yet their labeling efficiency toward bona fide small EVs (sEVs) remains poorly defined. Using a serum-free HEK293F system that produces endogenously fluorescent protein-tagged sEVs (sEVs-FPT) as an unambiguous reference while minimizing interference from dye-binding non-vesicular particles, we reassessed this efficiency with two orthogonal methods: nanoflow cytometry and fluorescence microscopy. PKH26, PKH67, and DiD labeled less than 0.5% of sEVs-FPT, regardless of vesicle heterogeneity. In vivo, dye-derived signals were far less abundant than tag-derived signals and failed to colocalize with them. Mechanistic evidence indicates that this failure stems from the inability of sEVs, as acellular structures, to actively internalize dye aggregates. These findings reveal a fundamental limitation of lipophilic dye-based EV tracking and underscore the need for orthogonally validated labeling strategies. One Sentence SummaryLipophilic dyes fail to label bona fide small extracellular vesicles, calling dye-based vesicle tracking into question.