Exosome labeling by lipophilic dye PKH26 results in significant increase in vesicle size

Abstract

Extracellular vesicles (EVs) are membrane vesicles secreted by cells and distributed widely in all biofluids. EVs can modulate the biological activities of cells in a paracrine or endocrine manner, in part by transferring their content, such as miRNA, following uptake in recipient cells. Fluorescent labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. Lipophilic fluorescent dyes such as those in the PKH family have been widely used for EV labelling. One concern with the use of lipophilic dyes is an increase in the EV size. This size shift alone may undermine the validity of EVs tracing studies as small changes in the size of inorganic nanoparticles are known to affect their cellular uptake and biodistribution. Here, the possibility of minimizing the size shift of PKH labelled EVs was systematically studied by changing the labelling condition. Unfortunately, the size shift towards larger particles was observed in all the PKH labelling conditions, including those where the labelled EVs were below the fluorescent detection limit. As opposed to lipophilic dyes, no significant shift in the size of labelled EVs was detected with protein binding dyes. Since the size shifts identified in all the PKH labelling conditions are likely to affect the cellular uptake and biodistribution, PKH may not be a reliable technique for EVs tracking.

Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes

“…Large fluorescent dots were observed in some fractions containing positive PKH26-labeled sEV or control PKH26-labeled sEV-free medium (an unspecific diffuse fluorescence in F4), suggesting the presence of some contaminants (e.g., aggregates, lipoproteins, protein complexes, ribonucleoproteins). This effect could be a consequence of high-speed centrifugation of culture medium in combination with lipophilic dyes such as PKH, which induce the formation of artifacts with different sizes and morphologies that can be detected by fluorescence microscopy [58,87,88]. The specialized literature has also reported a multitude of contaminants in vesicles separated by multi-step methods [60,89,90].…”

Autologous Versatile Vesicles‐Incorporated Biomimetic Extracellular Matrix Induces Biomineralization

“…[ 43 ] In addition, the initial sizes of two vesicles were similar, thus the membrane fusion or intercalation concerns brought by lipophilic dyes could be eliminated. [ 44 ] Besides PKH26 (red) 3,30‐octadecyloxacarbacyanate perchlorate (DiO, cell membrane green fluorescent dye) and DAPI were used for cytomembrane and nucleus staining. [ 45–47 ] E‐EVs were greatly internalized by BMSCs after incubation for 2 h, while L‐EVs were rarely engulfed ( Figure A,B).…”

Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

“…EV dyes do not reliably correlate with small EV content and may even increase vesicle size. Contamination of mislabeled lipoproteins and protein content and dye aggregation contributed to false positives (39)(40)(41). Therefore, we fused CD63 with an eGFP to label the HEK293T EVs.…”