Quantitative assessment of lipophilic membrane dye‐based labelling of extracellular vesicles by nano‐flow cytometry

Abstract

Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40 nm in diameter by a laboratory‐built nano‐flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%–80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%–70% of PFP‐EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di‐8‐ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di‐8‐ANEPPS and 70%–100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self‐aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di‐8‐ANEPPS and DiI, and the effect of LMD self‐aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very‐low‐density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma‐EV labelling. Besides, it was discovered that DSPE‐PEG2000‐biotin can only label ∼50% of plasma‐EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP.

Extracellular vesicles promote proliferation in an animal model of regeneration

“…Since EVs exert their influence on gene expression and cell behaviors through direct interaction with target cells 59 , we next tested whether neoblasts could take up EVs. We enriched planarian EVs using PEG precipitation, labeled EVs with the lipophilic dye PKH67, which labels the membrane of EVs 60 , then incubated these labeled EVs with dissociated cells (Fig. 2).…”

Kaempferia parviflora Extracellular Vesicle Loaded with Clarithromycin for the Treatment of Helicobacter pylori Infection

“…55 Importantly, Dil can be used for studying biological event without being cytotoxic and interfering with their bioactivity. 56,57 The internalization of PDEVs by recipient cells involves three mechanisms: endocytosis, receptor-mediated cell signaling, and phagocytosis. Doxorubicin-loaded lemon PDEVs were shown to be internalized into the cytoplasm of ovarian adenocarcinomas through caveolinmediated endocytosis.…”

Extracellular vesicles as carriers of mRNA: Opportunities and challenges in diagnosis and treatment

“…To capture an entire population of EVs, multiple surface markers can be used, but this increases overall expense and is relatively low-throughput 33 . Lipophilic and other complete membrane dyes may be used, however, these may alter EV properties, decrease stability, or increase particle aggregation 34 , 35 . Flow cytometry technologies have been expanded to the EV field, however, unbound dye may limit signaling from EVs resulting in low output readings after normalization 36 .…”