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You searched for: EV180017 (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 Sample condition
Experiment number
  • Experiments differ in Sample condition
Experiment number
  • Experiments differ in Sample condition
Experiment number
  • Experiments differ in Sample condition
Experiment number
  • Experiments differ in Sample condition
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV180017 1/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 63%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
63% (96th 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
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63/ Flotillin-2/ TSG101/ MT1-MMP
non-EV: PMP70/ CytochromeC/ Calnexin/ Golgin245
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-2, TSG101, MT1-MMP
Not detected contaminants
PMP70, CytochromeC, Calnexin, Golgin245
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
131
EV concentration
Yes
Particle yield
4.95E+09 particles/million cells
EV180017 2/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 62%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (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
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Expressing fascin nanobody
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
142
EV concentration
Yes
Particle yield
2.25E+10 particles/million cells
EV180017 5/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 62%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (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
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Expressing cortactin nanobody (NTA domain)
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Proteomics database
No
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
133
EV concentration
Yes
Particle yield
1.16E+09 particles/million cells
EV180017 3/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 50%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (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
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Expressing cortactin nanobody (SH3 domain)
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
133
EV concentration
Yes
Particle yield
9.79E+09 particles/million cells
EV180017 4/5 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Ultrafiltration
Beghein E 2018 50%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (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
DG + dUC + Filtration + Ultrafiltration
Protein markers
EV: CD9/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Expressing gelsolin nanobody
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Fluorescent NTA
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
128
EV concentration
Yes
Particle yield
1.97E+09 particles/million cells
1 - 5 of 5
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV180017
species
Homo sapiens
sample type
Cell culture
cell type
MDAMB231
condition
Control condition
Expressing
fascin nanobody
Expressing
cortactin nanobody
(NTA domain)
Expressing
cortactin nanobody
(SH3 domain)
Expressing
gelsolin nanobody
isolation protocol
DG
dUC
Filtration
Ultrafiltration
DG
dUC
Filtration
Ultrafiltration
DG
dUC
Filtration
Ultrafiltration
DG
dUC
Filtration
Ultrafiltration
DG
dUC
Filtration
Ultrafiltration
case number
1
2
5
3
4
EV-METRIC %
63
62
62
50
50