Surface Profiling of Extracellular Vesicles from Plasma or Ascites Fluid Using DotScan Antibody Microarrays

Belov, Larissa; Hallal, Susannah; Matic, Kieran J.; Zhou, Jerry; Wissmueller, Sandra; Ahmed, Nuzhat; Tanjil, Sumaiya; Mulligan, Stephen P.; Best, O. Giles; Simpson, Richard J.; Christopherson, Richard I.

doi:10.1007/978-1-4939-7057-5_20

Cited by 6 publications

(6 citation statements)

References 90 publications

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“…In contrast, Jamaly et al recently used various anticoagulants to isolate MV and measure their total plasma concentrations by NTA without finding any significant differences in numbers [68]. Apart from total MV numbers, the isolation of MV from serum drawn into lithium heparin tubes seems to result in the aggregation of platelet-derived MV, with sticky vesicle pellets and a reduction in platelet MV numbers, thereby specifically affecting certain MV subpopulations [69,70]. In addition to the anticoagulant, other preanalytical factors such as centrifugation, temperature, freeze-thaw cycles, or agitation can influence MV in blood samples, which should be taken into account when setting up isolation protocols for clinical studies [66,67,71].…”

Section: Analysis Of MV In Peripheral Bloodmentioning

confidence: 99%

“…Since PPP is often prepared by centrifugation at 2500-3000 × g, this step might result in a significant loss of larger MV, which already pellet at this force [9]. It has been proposed that two centrifugation steps at 1500× g might be preferable for MV isolation [70]; however, more elaborate sorting methods might be required to specifically isolate larger EV from blood.…”

Section: Analysis Of MV In Peripheral Bloodmentioning

confidence: 99%

“…Moreover, especially low-abundance tumor MV might be concealed in the heterogeneous mixture of blood MV, which might necessitate the development of novel protocols for specific enrichment of tumor MV populations. One suggestion is to remove the platelet-derived MV prior to analysis [ 70 ] since they represent the largest MV population in blood. A protocol was recently published for isolating different subpopulations of small and large EV from solid tumor tissue, in this example from melanoma [ 122 , 123 ].…”

Section: MV As Cancer Biomarkersmentioning

confidence: 99%

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Microvesicles in Cancer: Small Size, Large Potential

Menck

Sivaloganathan

Bleckmann

et al. 2020

IJMS

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Extracellular vesicles (EV) are secreted by all cell types in a tumor and its microenvironment (TME), playing an essential role in intercellular communication and the establishment of a TME favorable for tumor invasion and metastasis. They encompass a variety of vesicle populations, among them the well-known endosomal-derived small exosomes (Exo), but also larger vesicles (diameter > 100 nm) that are shed directly from the plasma membrane, the so-called microvesicles (MV). Increasing evidence suggests that MV, although biologically different, share the tumor-promoting features of Exo in the TME. Due to their larger size, they can be readily harvested from patients’ blood and characterized by routine methods such as conventional flow cytometry, exploiting the plethora of molecules expressed on their surface. In this review, we summarize the current knowledge about the biology and the composition of MV, as well as their role within the TME. We highlight not only the challenges and potential of MV as novel biomarkers for cancer, but also discuss their possible use for therapeutic intervention.

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Section: Analysis Of MV In Peripheral Bloodmentioning

confidence: 99%

Section: Analysis Of MV In Peripheral Bloodmentioning

confidence: 99%

Section: MV As Cancer Biomarkersmentioning

confidence: 99%

See 1 more Smart Citation

Microvesicles in Cancer: Small Size, Large Potential

Menck

Sivaloganathan

Bleckmann

et al. 2020

IJMS

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“…Later on, the same group reported the development of an array with capacity of screening 60 surface antigens from blood plasma without prior processing or enrichment [138]. Another established array, initially developed for leukaemia cells (DotScan), has been successfully implemented on vesicles for phenotypic analysis by fluorescence and chemiluminescence-based immunoassays [139].…”

Section: Microarraysmentioning

confidence: 99%

Phenotypic analysis of extracellular vesicles: a review on the applications of fluorescence

Παναγοπούλου

Wark

Birch

et al. 2020

J of Extracellular Vesicle

104

105

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Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed.

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“…The current limit for reliable FAVS is ~100 nm (Momen-Heravi et al, 2012). An alternative would be magnetism activated vesicle sorting (MAVS), but this is not currently feasible either, because the size of the magnetic beads necessary for effective separation, which would have to be delivered into releasable vesicles to tag them, exceeds the diameter of the synaptic vesicle (Belov et al, 2016). To assess changes in the molecular composition of synaptic vesicles over time, I thus used a two-colour STED imaging approach on ultrathin slices.…”

Section: Experimental Approach To Determine Changes In Synaptic Vesic...mentioning

confidence: 99%

Long-Term Temporal Dynamics of Synaptic Vesicles

Sven¹

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Neurotransmission requires the release of neurotransmitters from synaptic vesicles. This occurs via fusion of the vesicle to the pre-synaptic membrane upon stimulation. However, not all synaptic vesicles are equally releasable, and it has long been debated why the majority of synaptic vesicles do not respond to physiological levels of stimulation. I demonstrate here, using live-cell antibody-tagging in rat hippocampal cultures, that only young synaptic vesicles are releasing neurotransmitter, and that they become more reluctant to release as they age.This inactivation of synaptic vesicles is not strictly an ageing-dependent process, but conditional upon vesicle usage. I report here that synaptic vesicles release ~260 times, on average, before becoming inactive, and that increasing usage frequency speeds up inactivation. The inactivation is caused by contamination of synaptic vesicles with the cell membrane protein SNAP25. SNAP25 can interact with the vesicle protein CSPα in ciscomplexes on the vesicle itself. This sequesters CSPα and prevents the formation of transcomplexes with SNAP25 on the cell membrane. This trans-interaction, however, promotes vesicle fusion to the cell membrane. The more often a vesicle has fused to the cell membrane, the higher its chance is to be contaminated with SNAP25, and the less competent it is for future rounds of release. The inactivation of ageing synaptic vesicles is presumably coupled to usage to remove potentially damaged synaptic vesicles from neurotransmission. This hypothesis is strengthened by the observation of endocytosis defects and neurite degeneration when aged vesicles are forced to release. I further provide several timing parameters for key events in the life of synaptic vesicles, which can serve as a framework towards a quantitative model of the synaptic vesicle life cycle.

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Surface Profiling of Extracellular Vesicles from Plasma or Ascites Fluid Using DotScan Antibody Microarrays

Cited by 6 publications

References 90 publications

Microvesicles in Cancer: Small Size, Large Potential

Microvesicles in Cancer: Small Size, Large Potential

Phenotypic analysis of extracellular vesicles: a review on the applications of fluorescence

Long-Term Temporal Dynamics of Synaptic Vesicles

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