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You searched for: EV120020 (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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV120020 1/2 Mus musculus NAY (d)(U)C Bobrie A 2012 33%

Study summary

Full title
All authors
Bobrie A, Colombo M, Krumeich S, Raposo G, Théry C
Journal
J Extracell Vesicles
Abstract
Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosom (show more...)Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome-derived vesicles. We propose that the most commonly used protocols for exosome preparations co-purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles. (hide)
EV-METRIC
33% (75th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD63/ MFGE8/ Alix/ HSP70/ CD9
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
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 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Characterization: Protein analysis
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD63/ CD9/ HSP70/ TSG101/ MFGE8
Detected contaminants
Cell organelle protein
ELISA
Antibody details provided?
No
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
EM
EM-type
immune EM
EM protein
CD9
Image type
Wide-field
EV120020 2/2 Mus musculus NAY (d)(U)C
DG
Bobrie A 2012 33%

Study summary

Full title
All authors
Bobrie A, Colombo M, Krumeich S, Raposo G, Théry C
Journal
J Extracell Vesicles
Abstract
Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosom (show more...)Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome-derived vesicles. We propose that the most commonly used protocols for exosome preparations co-purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles. (hide)
EV-METRIC
33% (75th 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. For the quantitative method, the reporting of measured EV concentration is expected.
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
NAY
Focus vesicles
exosomes
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
DG
Protein markers
EV: CD63/ CD9/ MFGE8
non-EV:
Proteomics
no
EV density (g/ml)
1.09-1.14;1.26-1.29
Show all info
Study aim
Technical
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
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 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Density gradient
Lowest density fraction
0.4
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ MFGE8
ELISA
Antibody details provided?
No
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
None
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV120020
species
Mus musculus
sample type
Cell culture
cell type
NAY
condition
NAY
separation protocol
(d)(U)C
(d)(U)C
DG
Exp. nr.
1
2
EV-METRIC %
33
33