Imaging flow cytometry for the characterization of extracellular vesicles

Lannigan, Joanne; Erdbruegger, Uta

doi:10.1016/j.ymeth.2016.09.018

Cited by 92 publications

(120 citation statements)

References 79 publications

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“…To confirm this finding in our system, the protein binding dye 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) was used to label exosomes. CFSE dyes are membrane permeable chemical compounds which covalently bind to proteins and fluoresce after esterification in the lumen of the exosome [39,40].…”

Section: Resultsmentioning

confidence: 99%

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

Dehghani

Flax

et al. 2019

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

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

Dehghani

Flax

et al. 2019

Preprint

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“…Our principal aim was to develop a standardised method for the isolation and characterisation of individual large EVs, which could be further developed for phenotyping large EVs from clinical samples. The value of the Imagestream to the field of EV characterisation has been explored elsewhere (14,15,19). However, reports focussing on the large EV populations; frequently discarded during small EV isolation protocols, are rarely present in the current literature.…”

Section: Discussionmentioning

confidence: 99%

“…EVs were acquired for 5 minutes at x 60 magnification using lasers as described ( Table 2) (Image stream, Amnis, Seattle, United States). The 60 x objective provides a Numerical Aperture of 0.9 enabling resolution of 0.3 µm 2 / pixel (14).…”

Section: Antibody Labellingmentioning

confidence: 99%

“…Equivalent Soluble Fluorochrome (MESF) values were calculated as previously described (14,15). Quantibrite PE beads (Cat: 340495.…”

Section: Mesf Calculation: To Enable Comparisons Between Experimentsmentioning

confidence: 99%

“…Serial dilutions of CMO labelled EVs showed a linear decrease in objects per ml (Figure 2 B i) with an increasing dilution factor, whilst the fluorescence intensity remained stable across the dilutions (Figure 2 B ii). To facilitate reproducible reporting across experiments and to offer a means to standardise EV measurements, we determined the minimum MESF value for CMO+ events which would distinguish between unstained and CMO labelled EVs using our staining protocol, as described elsewhere (14). Therefore, to set a standardised lower threshold of detection which could be used across replicate experiments we assigned a lower MESF threshold for CMO+ events of 50 (Example shown in Figure 2 C v).…”

Section: Evs Are Highly Heterogeneous and Differentially Express Ev Mmentioning

confidence: 99%

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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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Recent Progress in Isolation and Detection of Extracellular Vesicles for Cancer Diagnostics

Wang

Luo

Wang

2018

Adv Healthcare Materials

121

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Extracellular vesicles (EVs) are emerging as one of the many new and promising biomarkers for liquid biopsy of cancer due to their loading capability of some specific proteins and nucleic acids that are closely associated with cancer states. As such, the isolation and detection of cancer-derived EVs offer important information in noninvasive diagnosis of early-stage cancer and real-time monitoring of cancer development. In light of the importance of EVs, over the last decade, researchers have made remarkable innovations to advance the development of EV isolation and detection methods by taking advantage of microfluidics, biomolecule probes, nanomaterials, surface plasmon, optics, and so on. This review introduces the basic properties of EVs and common cancer-derived EV ingredients, and provides a comprehensive overview of EV isolation and detection strategies, with emphasis on liquid biopsies of EVs for cancer diagnostics.

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Imaging flow cytometry for the characterization of extracellular vesicles

Cited by 92 publications

References 79 publications

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

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

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

Recent Progress in Isolation and Detection of Extracellular Vesicles for Cancer Diagnostics

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