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You searched for: EV100023 (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
EV100023 1/1 Mus musculus Cell culture supernatant DG
dUC
Mathews JA 2010 44%

Study summary

Full title
All authors
Mathews JA, Gibb DR, Chen BH, Scherle P, Conrad DH
Journal
J Biol Chem
Abstract
The low affinity receptor for IgE, CD23, is the natural regulator of IgE synthesis, and understandin (show more...)The low affinity receptor for IgE, CD23, is the natural regulator of IgE synthesis, and understanding both the synthesis and the catabolism of CD23 are, thus, important issues. Membrane CD23 is cleaved by a disintegrin and metalloproteinase 10 (ADAM10) and this cleavage influences the ability of CD23 to regulate IgE. In contrast to the belief that cleavage is a cell surface event, endosomal neutralization with NH(4)Cl was found to dramatically reduce CD23 cleavage, suggesting that the majority of CD23 cleavage occurred subsequent to internalization in the endosomal pathway and not at the cell surface. In line with this, full-length CD23 was shown to be sorted in an ADAM10-dependent manner into exosomes. Greatly increased ADAM10-mediated CD23 cleavage was seen at endosomal pH. Additionally, the stalk region of CD23 was found to interact with ADAM10 and ADAM10 binding of CD23 was found to be protease independent. SPR analysis of the interaction indicated about a 10-fold increase in the R(max) at endosomal pH (pH 5.8) compared with pH 7.4, whereas the affinity of the interaction was not significantly changed. The R(max) change, combined with the increased cleavage at endosomal pH, indicates greater accessibility of the CD23 stalk region for ADAM10 at the lower pH. These results indicate a model where CD23 internalization results in ADAM10-dependent incorporation into exosomes, followed by partial cleavage of CD23 by ADAM10 prior to being released from the cell. The increased cleavage at endosomal pH also has implications for other ADAM10 substrates. (hide)
EV-METRIC
44% (80th 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
124.9 (pelleting) / 124.9 (washing)
Protein markers
EV: ADAM10
non-EV:
Proteomics
no
EV density (g/ml)
1.38-1.4
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
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
AH650
Pelleting: adjusted k-factor
124.9
Wash: volume per pellet (ml)
5
Wash: Rotor Type
AH650
Wash: adjusted k-factor
124.9
Density gradient
Only used for validation of main results
1
Density medium
Sucrose
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Rotor type
AH650
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
ADAM10
ELISA
Detected EV-associated proteins
ADAM10
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