Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

Dragovic, Rebecca; Gardiner, Chris; Brooks, Alexandra S.; Tannetta, Dionne; Ferguson, David J. P.; Hole, Patrick; Carr, Bob; Redman, Christopher W.G.; Harris, Adrian L.; Dobson, Peter J.; Harrison, Paul; Sargent, Ian L.

doi:10.1016/j.nano.2011.04.003

Cited by 1,130 publications

(1,081 citation statements)

References 21 publications

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“…7A). Established EV hydrodynamic diameter was within the range reported for human plasma EVs (73). Next, we evaluated the presence of EVs in the three fractions collected following centrifugation through a 30% sucrose cushion.…”

Section: Detection Of Particle Hydrodynamic Diameter In Ev Preparationsmentioning

confidence: 85%

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

Saá

Yakovleva

Castro

et al. 2014

Journal of Biological Chemistry

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Background: Prions can be transmitted by blood transfusion, but their origin and distribution in blood are unknown. Results: Prions were detected in plasma extracellular vesicles from preclinical and clinically sick mice. Conclusion: Prions associate with blood-circulating extracellular vesicles. Significance: These findings provide information about prion distribution in blood and set the groundwork for novel prion removal and disease diagnosis technologies.

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Section: Detection Of Particle Hydrodynamic Diameter In Ev Preparationsmentioning

confidence: 85%

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

Saá

Yakovleva

Castro

et al. 2014

Journal of Biological Chemistry

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“…After confirming the presence of CD63-eGFP labelled EVs by fluorescence microscopy, the particle concentration and their size distribution were analysed by NTA [42,50]. As depicted in Figure 2(a), analysis of the particles harvested per mL conditioned media (CM) revealed PEG concentration dependence for all PEG variants.…”

Section: Resultsmentioning

confidence: 99%

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Ludwig

Miroschedji

Doeppner

et al. 2018

J of Extracellular Vesicle

198

206

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Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

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“…9,71 Western blot analysis of endogenous or chimeric CD63 in isolated exosomes is used to measure exosomes, 9 as the signal correlates with nanoparticle concentration data (nanoparticles/ml) as confirmed by Nanopaticle Tracking Analysis (NTA) 72,73 (Supplementary Figure S2). In preliminary experiments, we probed WB of purified exosomes with another exosome marker (Lamp-1) and plasma membrane markers (CD45 and CD28), to exclude the presence of cell debris, apoptotic bodies and/or shedding vesicles (Supplementary Figure S2).…”

Section: Methodsmentioning

confidence: 92%

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

et al. 2015

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Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic. Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types. 1 ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process. 2,3 Two main pathways are involved in MVB maturation. 4,5 In addition to the ESCRT (endosomal complex required for traffic) proteins, 6 there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA), 7 ceramides 8 and diacylglycerol (DAG) 9 contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activationinduced cell death (AICD), antigen presentation and intercellular miRNA exchange. [10][11][12][13][14][15] The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins. 14,16-21 These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes. 22 DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG. 23,24 The secretory vesicle pathway involves several DAGc...

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Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis

Cited by 1,130 publications

References 21 publications

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

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