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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
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
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV190076 1/1 Homo sapiens Urine dUC Musante L 2020 78%

Study summary

Full title
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% (95th 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
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
dUC
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
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
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 adjustments
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: Particle analysis
NA
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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