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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190076	1/1	Homo sapiens	Urine	(d)(U)C	Musante L	2020	78%
Study summary Full title Rigorous characterization of urinary extracellular vesicles (uEVs) in the low centrifugation pellet - a neglected source for uEVs. All authors Musante L, Bontha SV, La Salvia S, Fernandez-Piñeros A, Lannigan J, Le TH, Mas V, Erdbrügger U Journal Sci Rep Abstract Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centri (show more...)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. (hide) EV-METRIC 78% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ Podocin/ Podocalyxin/ Collectrin/ IGFBP7/ CD9 non-EV: Calnexin/ Tamm-Horsfall protein/ Albumin/ Calreticulin Proteomics yes Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type FA-45-24-11 Pelleting: speed (g) 21130 Wash: volume per pellet (ml) 1.2 Wash: time (min) 30 Wash: Rotor Type FA-45-24-11 Wash: speed (g) 21130 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD9/ Podocalyxin/ Collectrin/ Podocin/ TSG101 Detected contaminants Calnexin/ Calreticulin/ Albumin/ Tamm-Horsfall protein Flow cytometry Type of Flow cytometry ImageStreamX Mark II Hardware adaptation to ~100nm EV's Imaging flow cytometry (IFCM) is a method combining flow cytometry with imaging. All signals are collected through microscope objectives and quantified based on images detected by charge coupled devic Detected EV-associated proteins Podocalyxin/ Collectrin/ IGFBP7 Proteomics database No Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 175 EV concentration Yes TRPS Report type Modus Reported size (nm) 173 EV concentration Yes EM EM-type Cryo-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV190076
species	Homo sapiens
sample type	Urine
condition	Control condition
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	78