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You searched for: EV100035 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV100035 1/1 Homo sapiens Cell culture supernatant dUC
Immunoaffinity (valid.)
Sucrose-DG
Buschow SI 2010 44%

Study summary

Full title
All authors
Buschow SI, van Balkom BW, Aalberts M, Heck AJ, Wauben M, Stoorvogel W
Journal
Immunol Cell Biol
Abstract
Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) car (show more...)Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHC II at lysosomally targeted multivesicular bodies, sorting of MHC II into exosomes does not rely on MHC II ubiquitination. In search for proteins that drive the incorporation of MHC II into exosomes or functionally discriminate exosomal from plasma membrane MHC II, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHC II from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3?, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHC II. (hide)
EV-METRIC
44% (81st 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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Immunoaffinity (valid.) + Sucrose-DG
Adj. k-factor
362.8 (pelleting)
Protein markers
EV: CD81/ MHC2
non-EV: None
Proteomics
yes
EV density (g/ml)
1.150
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
362.8
Density gradient
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
CD81/ MHC2
ELISA
Detected EV-associated proteins
MHC2
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