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You searched for: EV120004 (EV-TRACK ID)

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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 ultracentrifugation
    • 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
EV120004 1/1 Homo sapiens Cell culture supernatant DG
dUC
de Jong OG 2012 67%

Study summary

Full title
All authors
de Jong OG, Verhaar MC, Chen Y, Vader P, Gremmels H, Posthuma G, Schiffelers RM, Gucek M, van Balkom BW
Journal
J Extracell Vesicles
Abstract
BACKGROUND: The healthy vascular endothelium, which forms the barrier between blood and the surround (show more...)BACKGROUND: The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis. It has been suggested that exosomes protect their target cells from cellular stress through the transfer of RNA and proteins. We hypothesized that endothelium-derived exosomes are involved in the endothelial response to cellular stress, and that exosome RNA and protein content reflect the effects of cellular stress induced by hypoxia, inflammation or hyperglycemia. METHODS: We exposed cultured endothelial cells to different types of cellular stress (hypoxia, TNF-?-induced activation, high glucose and mannose concentrations) and compared mRNA and protein content of exosomes produced by these cells by microarray analysis and a quantitative proteomics approach. RESULTS: We identified 1,354 proteins and 1,992 mRNAs in endothelial cell-derived exosomes. Several proteins and mRNAs showed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis. CONCLUSION: Our data show that hypoxia and endothelial activation are reflected in RNA and protein exosome composition, and that exposure to high sugar concentrations alters exosome protein composition only to a minor extend, and does not affect exosome RNA composition. (hide)
EV-METRIC
67% (94th 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
Cell culture supernatant
Sample origin
DNF
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Adj. k-factor
256 (pelleting)
Protein markers
EV: HSP70/ Beta-actin/ Flotilin1/ CD63/ ICAM1
non-EV:
Proteomics
yes
EV density (g/ml)
1.09-1.12
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW32;SW60
Pelleting: adjusted k-factor
256.0
Density gradient
Only used for validation of main results
1
Density medium
Sucrose
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ HSP70/ Beta-actin/ ICAM1
ELISA
Detected EV-associated proteins
Beta-actin/ ICAM1
Characterization: Particle analysis
NTA
EM
EM-type
immune EM
Proteïns
CD63
Image type
Wide-field
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