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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
EV120093 1/1 Mus musculus Cell culture supernatant DG
dUC
van der Vlist EJ 2012 43%

Study summary

Full title
All authors
van der Vlist EJ, Arkesteijn GJ, van de Lest CH, Stoorvogel W, Nolte-'t Hoen EN, Wauben MH
Journal
J Extracell Vesicles
Abstract
Many cell types release nanosized vesicles derived from endosomal compartments (exosomes) or the pla (show more...)Many cell types release nanosized vesicles derived from endosomal compartments (exosomes) or the plasma membrane. Vesicles actively released by CD4(+) T cells have immune-modulatory characteristics. Using our recently developed high-resolution flow cytometry-based method for the analysis of individual nanosized vesicles, we here investigated how T cell receptor (TCR)-triggering and co-stimulatory signals influence the quantity and characteristics of nanosized vesicles released by CD4(+) T cells. We found that the number of released nanosized vesicles within the buoyant density range characteristic for exosomes (1.10-1.19 g/ml) was increased by TCR-triggering and that additional co-stimulatory signals had a potentiating effect on vesicle release. However, the increase in the number of released vesicles varied substantially between density fractions within the 1.10-1.19 g/ml range and was highest for the vesicle populations in 1.14 and 1.17 g/ml fractions. Heterogeneity was also observed within the individual density fractions. Based on lipid bilayer fluorescent labelling intensity and light scattering, 3 distinct vesicle subpopulations were identified. One vesicle subpopulation increased significantly more upon T cell activation than the other subpopulations, and this was dependent on high levels of co-stimulation. These data show that T cells release a heterogeneous population of nanosized vesicles and indicate that T cells differentially regulate the release of distinct vesicle subpopulations depending on their activation status. (hide)
EV-METRIC
43% (76th 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
Nano(-sized) vesicles
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
276.6 (pelleting)
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.1-1.19
Show all info
Study aim
Biogenesis/Sorting
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)
65
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
276.6
Density gradient
Density medium
Sucrose
Lowest density fraction
0.4
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Particle analysis
NTA
Particle analysis: flow cytometry
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