An accurate, precise method for general labeling of extracellular vesicles

Gray, Warren D.; Mitchell, Adam; Searles, Charles

doi:10.1016/j.mex.2015.08.002

Cited by 122 publications

(122 citation statements)

References 13 publications

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“…2a). Calcein is a high-sensitivity marker that emits fluorescence when internalized into the intact EVs [26]. At 6 weeks, elevated circulating EV number was associated with increased abundance of cleaved/active caspase 3 in epididymal AT of DIO mice compared to chow fed controls (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress

et al. 2016

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We recently reported that stressed adipocytes release extracellular vesicles (EVs) that act as “find me” signals to promote macrophage migration and activation. In this study we performed a comprehensive characterization of stressed adipocyte-derived EVs, assessing their antigenic composition, lipidomics, and RNA profiles. Perilipin A was identified as one of the adipose-specific proteins and studied as a potential novel biomarker to detect adipocyte-derived EVs in circulation. Circulating EVs were significantly increased in mice with diet-induced obesity (DIO) and in obese humans with Metabolic Syndrome compared to lean controls. This increase was associated with decreased glucose tolerance in the DIO mice and metabolic dysfunction, elevated insulin and homeostatic model assessment of insulin resistance (HOMA-IR), in the obese humans. EVs from both DIO mice and obese humans were enriched in perilipin A, a central gatekeeper of the adipocyte lipid storehouse and a marker of adipocyte differentiation. In obese humans, circulating levels of EVs enriched in perilipin A were dynamic, decreasing 35% (p<0.05) after a 3-month reduced calorie diet intervention. This translational study provides an extensive characterization of adipocyte-derived EVs. The findings identify perilipin A as a novel biomarker of circulating EVs of adipocyte origin and support the development of circulating perilipin A-positive EVs as indicators of adipose tissue health.

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Section: Resultsmentioning

confidence: 99%

Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress

et al. 2016

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“…From crude EV samples, intact EVs were labelled with calcein violet 450 AM (eBioscience; 65-0854-39) as previously described (Gray et al, 2015). Detergent treated negative controls were mixed with 0.05% Triton X-100 (Sigma: T8787).…”

Section: Flow Cytometrymentioning

confidence: 99%

In vivo characterisation of endogenous cardiovascular extracellular vesicles in larval and adult zebrafish

Scott

Ballesteros

Bradshaw

et al. 2019

Preprint

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Ischaemic cardiac injury results in a complex coordinated cellular response that enables effective damage control and recovery. Intercellular communication is integral to the coordination of these repair responses in complex tissue contexts. Extracellular vesicles (EVs) facilitate molecular transport across extracellular space, allowing local and systemic signaling during homeostasis and in disease. Extensive in vitro studies have described the biogenesis, cargos and functional roles of EV populations but the characterisation of endogenously produced EVs in vivo is still in its infancy. Here we use larval and adult transgenic zebrafish to characterize endogenous cardiovascular EVs. Using a membranetethered fluorophore reporter system, cell-type specific EVs can be tracked in vivo by high spatiotemporal resolution light sheet imaging and modified flow cytometry methods allows these EVs to be extracted and assessed ex vivo. Importantly, we show that ischaemic cardiac injury models dynamically alter EV production during repair.

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“…Another example is the use of calcein AM (an acetoxymethyl derivate of the fluorescent molecule calcein) that is a very good cytoplasmic fluorescent dye, since it attains high fluorescence intensities and exhibits an acceptable persistent labeling, given that it does not covalently link to intracellular molecules [33]. Calcein-labeling strategy is based on a membrane-permeant molecule that is nonfluorescent until it is activated by intra-vesicular enzymes [42]. Upon hydrolysis of the acetoxymethyl ester moieties by esterases, calcein Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases becomes highly membrane impermeable [42].…”

Section: Membrane Permeable Fluorescent Dyesmentioning

confidence: 99%

“…Calcein-labeling strategy is based on a membrane-permeant molecule that is nonfluorescent until it is activated by intra-vesicular enzymes [42]. Upon hydrolysis of the acetoxymethyl ester moieties by esterases, calcein Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases becomes highly membrane impermeable [42]. The detection of calcein-labeled exosomes through flow cytometry has been already described and its use in some experiments has also been reported [43,44].…”

Section: Membrane Permeable Fluorescent Dyesmentioning

confidence: 99%

Animal Models in Exosomes Research: What the Future Holds

Adem¹,

Melo²

2017

Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

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Exosomes have been implicated in a wide range of pathological and nonpathological processes. Research on tumor-derived exosomes uncovered their role on major processes associated with disease progression. Uncontrolled cellular proliferation resulting in tumor growth, metastatic dissemination and modulation of the immune response, are only a few of the central pathological processes in which tumor-derived exosomes have been implicated. These in vivo studies rely on the administration of purified labeled exosomes from cell culture supernatants into circulation of animals or injections of genetically engineered cells that produce labeled exosomes. However, it is not clear that current available techniques actually translate the in vivo implications of exosomes in several biological processes. The variations seen when using different exosomes cell sources, the total amount of exosomes injected in mice and their route of administration as well as the fact that most studies are performed in immunodeficient animals, shows the difficulty to achieve conclusions which are biologically significant. Genetically engineered mouse models (GEMM) could be a promising approach to address the current technical limitations allowing tracing tumor-derived exosomes in a living organism. These models could enhance greatly our knowledge about exosomes in different fields of research, namely cancer.

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An accurate, precise method for general labeling of extracellular vesicles

Abstract: Graphical abstract

Cited by 122 publications

References 13 publications

Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress

Circulating adipocyte-derived extracellular vesicles are novel markers of metabolic stress

In vivo characterisation of endogenous cardiovascular extracellular vesicles in larval and adult zebrafish

Animal Models in Exosomes Research: What the Future Holds

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