Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis

Pasalic, Leonardo; Williams, Rebekka; Siupa, Agnieszka; Campbell, Heather; Henderson, Michelle J.; Chen, Vivien

doi:10.1016/j.nano.2015.12.370

Cited by 30 publications

(18 citation statements)

References 26 publications

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“…Inter-user and inter-experiment variability is also a known concern with NTA, particularly among inexperienced users, though steps are being taken to improve this ( 45 – 48 ). The merits of FCM for particle enumeration over other techniques such as NTA are beyond our scope and described elsewhere ( 14 , 49 , 50 ). What is noteworthy however, is the capacity for iFCM to enumerate sEV subsets specifically, rather than vesicles taken together, or indeed merely the vesicle-sized particles measured by some other techniques.…”

Section: Discussionmentioning

confidence: 99%

Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry

et al. 2018

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Extracellular vesicles (EVs) are responsible for a multitude of physiological functions, including immunomodulation. A heterogenous mixture of small EV (sEV) subsets, including putative exosomes, is derived when commonly used “exosome” isolation techniques are employed. Subset diversity relates in part to their different intracellular origins, and can be associated with distinct functional properties. Recent progress in the EV field has enabled the categorization of such subsets based on their surface composition. For the first time, we combine such emerging subset-specific markers with advanced imaging flow cytometry (iFCM) to perform high-throughput, multiparametric, vesicle-by-vesicle characterization, and functional assessment of specific small EV subsets, and exosomes in particular. The approach allows researchers to address three important applications. First, it is known that different isolation techniques result in the divergent recovery of particular vesicle subsets. Taking three commonly used “exosome” isolation techniques as test cases (ultracentrifugation, size-exclusion chromatography, and polymer-based precipitation), the capacity for convenient and accurate isolate compositional analysis by iFCM is demonstrated. The approach was able to corroborate and to quantify the known skewing of subtype recovery among different isolation approaches. Second, exosomes are a particularly widely studied EV subset. Applying exosome-specific markers to samples collected from an optimal clinical transplantation model, we verify the capacity for iFCM to detect exosomes in circulation, to establish their tissue of origin, and to provide insights as to their functional immunological potential. Finally, we describe a technique for establishing whether the transfer of a molecule of interest to a target cell is exosomally mediated. In so doing, we highlight the approach’s utility in assessing the functional impact of circulating exosomes and in identifying their targets. In conclusion, we set out a new methodological approach by which small extracellular vesicle subsets, exosomes in particular, can be conveniently and comprehensively investigated, thereby offering novel phenotypic and functional insights.

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Section: Discussionmentioning

confidence: 99%

Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry

et al. 2018

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“…It has been demonstrated that enumeration of EVs using a fluorescent triggering threshold, employing a fluorescent membrane intercalating dye, offers higher sensitivity than using scatter triggering threshold (Nolte-'t Hoen et al, 2012 ; van der Vlist et al, 2012 ; Arraud et al, 2015a , b ; Pasalic et al, 2016 ; Stoner et al, 2016 ). This method of detection requires a wash step, or titering of the dye, to ensure minimal quantities of residual dye.…”

Section: Fluorescence Detection Standardizationmentioning

confidence: 99%

Extracellular Vesicle Flow Cytometry Analysis and Standardization

Welsh

Holloway

Wilkinson

et al. 2017

Front. Cell Dev. Biol.

108

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The term extracellular vesicles (EVs) describes membranous vesicles derived from cells, ranging in diameter from 30 to 1,000 nm with the majority thought to be in the region of 100–150 nm. Due to their small diameter and complex and variable composition, conventional techniques have struggled to accurately count and phenotype EVs. Currently, EV characterization using high-resolution flow cytometry is the most promising method when compared to other currently available techniques, due to it being a high-throughput, single particle, multi-parameter analysis technique capable of analyzing a large range of particle diameters. Whilst high resolution flow cytometry promises detection of the full EV diameter range, standardization of light scattering and fluorescence data between different flow cytometers remains an problem. In this mini review, we will discuss the advances in high-resolution flow cytometry development and future direction of EV scatter and fluorescence standardization. Standardization and therefore reproducibility between research groups and instrumentation is lacking, hindering the validation of EVs use as diagnostic biomarkers and therapeutics.

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“…Infectious agents [20,21], subcellular components ( e.g. , plasma membrane) [22,23] can also be tracked, as can the fate and bioactivity of cell-derived vesicles [24–26]. Combining fluorescent cell tracking dyes with stably expressed genetic markers has become increasingly common as the spectral choices available for probe types have increased.…”

Section: Introductionmentioning

confidence: 99%

Monitoring Cell Proliferation by Dye Dilution: Considerations for Probe Selection

Tario

Conway

Muirhead³

et al. 2017

Methods in Molecular Biology

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In the third edition of this series, we described protocols for labeling cell populations with tracking dyes, and addressed issues to be considered when combining two different tracking dyes with other phenotypic and viability probes for the assessment of cytotoxic effector activity and regulatory T cell functions. We summarized key characteristics of and differences between general protein and membrane labeling dyes, discussed determination of optimal staining concentrations, and provided detailed labeling protocols for both dye types. Examples of the advantages of two color cell tracking were provided in the form of protocols for: (a) independent enumeration of viable effector and target cells in a direct cytotoxicity assay; and (b) an in vitro suppression assay for simultaneous proliferation monitoring of effector and regulatory T cells. The number of commercially available fluorescent cell tracking dyes has expanded significantly since the last edition, with new suppliers and/or new spectral properties being added at least annually. In this fourth edition, we describe evaluations to be performed by the supplier and/or user when characterizing a new cell tracking dye and by the user when selecting one for use in multicolor proliferation monitoring. These include methods for: Assessment of the dye’s spectral profile on the laboratory’s flow cytometer(s) to optimize compatibility with other employed fluorochromes and minimize compensation problems;Evaluating the effect of labeling on cell growth rate;Testing the fidelity with which dye dilution reports cell division;Determining the maximum number of generations to be included when using dye dilution profiles to estimate fold population expansion or frequency of responder cells; andVerifying that relevant cell functions (e.g., effector activity) remain unaltered by tracking dye labeling.

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Enumeration of extracellular vesicles by a new improved flow cytometric method is comparable to fluorescence mode nanoparticle tracking analysis

Cited by 30 publications

References 26 publications

Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry

Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry

Extracellular Vesicle Flow Cytometry Analysis and Standardization

Monitoring Cell Proliferation by Dye Dilution: Considerations for Probe Selection

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