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You searched for: EV170061 (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
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV170061 1/3 Homo sapiens Cell culture supernatant dUC
Filtration
SEC
Ultrafiltration
Benedikter BJ 2017 62%

Study summary

Full title
All authors
Benedikter BJ, Bouwman FG, Vajen T, Heinzmann ACA, Grauls G, Mariman EC, Wouters EFM, Savelkoul PH, Lopez-Iglesias C, Koenen RR, Rohde GGU, Stassen FRM
Journal
J Cell Sci
Abstract
Appropriate isolation methods are essential for unravelling the relative contribution of extracellul (show more...)Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules. (hide)
EV-METRIC
62% (95th 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
Focus vesicles
extracellular vesicle
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 + Filtration + SEC + Ultrafiltration
Protein markers
EV: CD63/ CD81/ MFGE8/ CD9
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, MFGE8
Flow cytometry specific beads
Selected surface protein(s)
CD9, CD63, CD81
Proteomics database
No
Characterization: Particle analysis
TRPS
Report type
Median
Reported size (nm)
61.9
EV concentration
Yes
Particle yield
3.00E+01
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
25-200
EV170061 2/3 Homo sapiens Cell culture supernatant dUC Benedikter BJ 2017 50%

Study summary

Full title
All authors
Benedikter BJ, Bouwman FG, Vajen T, Heinzmann ACA, Grauls G, Mariman EC, Wouters EFM, Savelkoul PH, Lopez-Iglesias C, Koenen RR, Rohde GGU, Stassen FRM
Journal
J Cell Sci
Abstract
Appropriate isolation methods are essential for unravelling the relative contribution of extracellul (show more...)Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules. (hide)
EV-METRIC
50% (89th 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
Focus vesicles
extracellular vesicle
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
Adj. k-factor
133.2 (pelleting) / 48.17 (washing)
Protein markers
EV: CD63/ CD81
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
117734
Pelleting: adjusted k-factor
133.2
Wash: time (min)
150
Wash: Rotor Type
NVT 90
Wash: speed (g)
110656
Wash: adjusted k-factor
48.17
Characterization: Protein analysis
Protein Concentration Method
Bradford
Flow cytometry specific beads
Selected surface protein(s)
CD63
Proteomics database
No
Characterization: Particle analysis
TRPS
EV concentration
Yes
Particle yield
2.00E+00
EV170061 3/3 Homo sapiens Cell culture supernatant dUC Benedikter BJ 2017 50%

Study summary

Full title
All authors
Benedikter BJ, Bouwman FG, Vajen T, Heinzmann ACA, Grauls G, Mariman EC, Wouters EFM, Savelkoul PH, Lopez-Iglesias C, Koenen RR, Rohde GGU, Stassen FRM
Journal
J Cell Sci
Abstract
Appropriate isolation methods are essential for unravelling the relative contribution of extracellul (show more...)Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules. (hide)
EV-METRIC
50% (89th 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
Focus vesicles
extracellular vesicle
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
Adj. k-factor
133.2 (pelleting)
Protein markers
EV: CD63/ CD81
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
BEAS2B
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
117734
Pelleting: adjusted k-factor
133.2
Characterization: Protein analysis
Protein Concentration Method
Bradford
Flow cytometry specific beads
Selected surface protein(s)
CD63
Proteomics database
No
Characterization: Particle analysis
TRPS
Report type
Median
Reported size (nm)
74.6
EV concentration
Yes
Particle yield
1.50E+01
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
25-200
1 - 3 of 3
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV170061
species
Homo sapiens
sample type
Cell culture
cell type
BEAS2B
condition
Control condition
isolation protocol
dUC
Filtration
SEC
Ultrafiltration
dUC
dUC
case number
1
2
3
EV-METRIC %
62
50
50