Dataset Information

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs.

ABSTRACT: Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (?9.0?mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. Cryo-Transmission Electron Microscopy (?3.0?mL urine) provided evidence of a heterogeneous population of EVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (?4.5?mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5?mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single EV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0?mL of urine respectively. This work characterizes a neglected source of uEVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of uEV analysis such as autofluorescence of podocyte origin.

SUBMITTER: Musante L

PROVIDER: S-EPMC7048852 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs.

Musante Luca L Bontha Sai Vineela SV La Salvia Sabrina S Fernandez-Piñeros Angela A Lannigan Joanne J Le Thu H TH Mas Valeria V Erdbrügger Uta U

Scientific reports 20200228 1

Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine req ...[more]

PMID: 32111925

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Project description:Extracellular vesicle (EV) research increasingly demands for quantitative characterisation at the single vesicle level to address heterogeneity and complexity of EV subpopulations. Emerging, commercialised technologies for single EV analysis based on, for example, imaging flow cytometry or imaging after capture on chips generally require dedicated instrumentation and proprietary software not readily accessible to every lab. This limits their implementation for routine EV characterisation in the rapidly growing EV field. We and others have shown that single vesicles can be detected as light diffraction limited fluorescent spots using standard confocal and widefield fluorescence microscopes. Advancing this simple strategy into a process for routine EV quantitation, we developed 'EVAnalyzer', an ImageJ/Fiji (Fiji is just ImageJ) plugin for automated, quantitative single vesicle analysis from imaging data. Using EVAnalyzer, we established a robust protocol for capture, (immuno-)labelling and fluorescent imaging of EVs. To exemplify the application scope, the process was optimised and systematically tested for (i) quantification of EV subpopulations, (ii) validation of EV labelling reagents, (iii) in situ determination of antibody specificity, sensitivity and species cross-reactivity for EV markers and (iv) optimisation of genetic EV engineering. Additionally, we show that the process can be applied to synthetic nanoparticles, allowing to determine siRNA encapsulation efficiencies of lipid-based nanoparticles (LNPs) and protein loading of SiO2 nanoparticles. EVAnalyzer further provides a pipeline for automated quantification of cell uptake at the single cell-single vesicle level, thereby enabling high content EV cell uptake assays and plate-based screens. Notably, the entire procedure from sample preparation to the final data output is entirely based on standard reagents, materials, laboratory equipment and open access software. In summary, we show that EVAnalyzer enables rigorous characterisation of EVs with generally accessible tools. Since we further provide the plugin as open-source code, we expect EVAnalyzer to not only be a resource of immediate impact, but an open innovation platform for the EV and nanoparticle research communities.

Dataset Information

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs.

Publications

Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs.

Similar Datasets

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