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

Simonsen, Jens B.

doi:10.1080/20013078.2019.1582237

Cited by 120 publications

(104 citation statements)

References 27 publications

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“…These compounds are lipid‐like molecules with fluorescent head groups and long aliphatic tails capable of inserting into the vesicle membrane and have become of extensive use for fluorescence‐based studies of EVs [16–19]. The major drawback of lipophilic tracers, however, is that they are not EV specific and indiscriminately stain all lipid‐containing particles and vesicles within the sample [29,30]. Likewise, being lipophilic, they can also redistribute from the EVs to the cellular membranes.…”

Section: Introductionmentioning

confidence: 99%

Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Corso

Heusermann

Trojer

et al. 2019

J of Extracellular Vesicle

132

137

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Section: Introductionmentioning

confidence: 99%

Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Corso

Heusermann

Trojer

et al. 2019

J of Extracellular Vesicle

132

137

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“…Though lipid‐anchored fluorophores were reported to label non‐EV serum components which might decrease the combination efficiency with EVs, PKH26 is still a common choice to track EVs to qualitatively investigate the internalization behavior by cells. [ 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.…”

Section: Resultsmentioning

confidence: 99%

Autologous Versatile Vesicles‐Incorporated Biomimetic Extracellular Matrix Induces Biomineralization

Wei

Shi

Zhang

et al. 2020

Adv Funct Materials

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Restoring extracellular matrix (ECM) with supportive and osteoinductive abilities is of great significance for bone tissue regeneration. Current approaches involving cell-based scaffolds or nanoparticle-modified biomimic-ECM have been met with additional biosafety concerns. Herein, the natural biomineralization process is first analyzed and is found that mesenchymal stem cells-derived extracellular vesicles (EVs) from early and late stages of osteoinduction play different roles during the mineralization process. The functional EVs hierarchically with blood-derived autohydrogel (AH) are then incorporated to form an osteoinductive biomimetic extracellular matrix (BECM). The alkaline phosphatase-rich EVs are released from the outer layers to induce osteoblast differentiation during early stages. Thereafter, as the degradation of AH occurred, calcium/phosphorus (Ca/P)rich EVs are liberated to promote the nucleation of extracellular mineral crystals. Additionally, BECM contains considerable collagen fibrils that provide additional nucleation sites for crystallites deposition, thus reaching self-mineralization in situ. In conclusion, this research provides a promising, versatile mineralization-instructive platform to tackle the challenges faced in bone-tissue engineering. Scheme 1. Illustration of the BECM induced osteogenesis and biomineralization. In the early stage of mineralization, BECM secreted ALP-rich E-EVs. The E-EVs moved to intracellular mitochondria and provided ALP for phosphate metabolism, contributing to the formation of Ca/P-rich vesicles. Later, with the degradation of BECM, Ca/P-rich L-EVs were released gradually. The L-EVs aligned to extracellular collagen and promoted the nucleation of extracellular mineral crystals. Besides, L-EVs inside BECM aggregate around the internally collagen fibrils and initiated in situ mineralization. Finally, with the complete degradation of BECM, the internal mineralized components were released to the extracellular matrix, promoting the deposition formation together to realize an ideal self-mineralization process for bone tissue regeneration.www.afm-journal.de www.advancedsciencenews.com (3 of 15)

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“…By using in vitro cultures, we were able to use the ideal conditions to generate EVs and optimise experiments. Specifically, by culturing in serum free media, we eliminated contamination from bovine EVs present in FBS (16) and subsequent false positive fluorescence signals from serum lipoproteins (17). Nevertheless, harvesting large EVs from any source presents challenges as cells or cell debris, including intracellular vesicles released due to parent cell membrane rupture from early centrifugation steps, can contaminate the subsequent EV pellets.…”

Section: Discussionmentioning

confidence: 99%

“…Previous reports have identified that lipid dye aggregates can mimic EVs when using fluorescence lipid markers (17). We used Triton-X 100 treatment (18)…”

Section: Evs Are Highly Heterogeneous and Differentially Express Ev Mmentioning

confidence: 99%

Large extracellular vesicles can be characterised by multiplex labelling using imaging flow cytometry

Johnson

Banyard

Smith

et al. 2020

Preprint

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Extracellular vesicles (EVs) are heterogeneous in size (30nm-10µm), content (lipid, RNA, DNA, protein) and potential function(s). Many isolation techniques routinely discard the large EVs at the early stages of small EV or exosome isolation protocols. We describe here a standardised method to isolate large EVs and examine EV marker expression and diameter using imaging flow cytometry. Methods: We describe step-wise isolation and characterisation of a subset of large EVs from the medulloblastoma cell line UW228-2 assessed by fluorescent light microscopy, transmission electron microscopy (TEM) and TRPS. Viability of parent cells was assessed by Annexin V exposure by flow cytometry. Imaging flow cytometry (Imagestream Mark II) identified EVs by direct fluorescent membrane labelling with Cell Mask Orange (CMO) in conjunction with EV markers. A stringent gating algorithm based on side scatter and fluorescence intensity was applied and expression of EV markers CD63, CD9 and LAMP 1 assessed.Results: UW228-2 cells prolifically release EVs of up to 6 µm. We show that the Imagestream Mark II imaging flow cytometer allows robust and reproducible analysis of large EVs, including assessment of diameter. We also demonstrate a correlation between increasing EV size and co-expression of markers screened. Conclusions:We have developed a labelling and stringent gating strategy which is able to explore EV marker expression (CD63, CD9 and LAMP1) on individual EVs within a widely heterogeneous population. Taken together data presented here strongly support the value Johnson et al, 2020 3

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

Cited by 120 publications

References 27 publications

Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Systematic characterization of extracellular vesicle sorting domains and quantification at the single molecule – single vesicle level by fluorescence correlation spectroscopy and single particle imaging

Autologous Versatile Vesicles‐Incorporated Biomimetic Extracellular Matrix Induces Biomineralization

Large extracellular vesicles can be characterised by multiplex labelling using imaging flow cytometry

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