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You searched for: EV140203 (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
EV140203 1/1 Mus musculus Cell culture supernatant DG
dUC
Filtration
Padro CJ 2014 44%

Study summary

Full title
All authors
Padro CJ, Shawler TM, Gormley MG, Sanders VM
Journal
J Immunol
Abstract
Soluble CD23 plays a role in the positive regulation of an IgE response. Engagement of the ?2 adrene (show more...)Soluble CD23 plays a role in the positive regulation of an IgE response. Engagement of the ?2 adrenergic receptor (?2AR) on a B cell is known to enhance the level of both soluble CD23 and IgE, although the mechanism by which this occurs is not completely understood. In this study, we report that, in comparison with a CD40 ligand/IL-4-primed murine B cell alone, ?2AR engagement on a primed B cell increased gene expression of a disintegrin and metalloproteinase (ADAM)10, which is the primary sheddase of CD23, as well as protein expression of both CD23 and ADAM10, in a protein kinase A- and p38 MAPK-dependent manner, and promoted the localization of these proteins to exosomes as early as 2 d after priming, as determined by both Western blot and flow cytometry and confirmed by electron microscopy. In comparison with isolated exosomes released from primed B cells alone, the transfer of exosomes released from ?2AR agonist-exposed primed B cells to cultures of recipient primed B cells resulted in an increase in the level of IgE produced per cell, without affecting the number of cells producing IgE, as determined by ELISPOT. These effects still occurred when a ?2AR antagonist was added along with the transfer to block residual agonist, and they failed to occur when exosomes were isolated from ?2AR-deficient B cells. These findings suggest that the mechanism responsible for mediating the ?2AR-induced increase in IgE involves a shuttling of the ?2AR-induced increase in CD23 and ADAM10 proteins to exosomes that subsequently mediate an increase in IgE. (hide)
EV-METRIC
44% (79th 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 + Filtration
Adj. k-factor
255.8 (pelleting)
Protein markers
EV: ADAM10/ CD63
non-EV:
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW41
Pelleting: adjusted k-factor
255.8
Density gradient
Only used for validation of main results
1
Density medium
Iodixanol
Lowest density fraction
1.00g/L
Highest density fraction
1.25g/L
Orientation
Top-down
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ ADAM10
ELISA
Detected EV-associated proteins
ADAM10
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
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