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You searched for: EV190075 (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
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV190075 1/3 Homo sapiens Cell culture supernatant Filtration
Total Exosome Isolation
UF
Thomas E. Whittaker 2020 50%

Study summary

Full title
All authors
Thomas E. Whittaker, Anika Nagelkerke , Valeria Nele , Ulrike Kauscher & Molly M. Stevens
Journal
J Extracell Vesicles
Abstract
It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can (show more...)It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs. (hide)
EV-METRIC
50% (83rd 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
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
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
Filtration + Total Exosome Isolation + UF
Protein markers
EV: / CD81/ CD63/ CD9
non-EV: VEGF
Proteomics
no
Show all info
Study aim
Function/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
mesenchymal stem cells
EV-harvesting Medium
Serum free medium
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Ultra filtration
Cut-off size (kDa)
3
Membrane type
Regenerated cellulose
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
ELISA
Detected EV-associated proteins
Not detected EV-associated proteins
Detected contaminants
VEGF
Not detected contaminants
CD63/ CD81/ CD9
Other 1
Dot Blot
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
115.8
EV concentration
Yes
EV190075 2/3 Homo sapiens Cell culture supernatant Filtration
SEC
Size-exclusion chromatography (non-commercial)
UF
Thomas E. Whittaker 2020 50%

Study summary

Full title
All authors
Thomas E. Whittaker, Anika Nagelkerke , Valeria Nele , Ulrike Kauscher & Molly M. Stevens
Journal
J Extracell Vesicles
Abstract
It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can (show more...)It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs. (hide)
EV-METRIC
50% (83rd 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
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
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
Filtration + SEC + Size-exclusion chromatography (non-commercial) + UF
Protein markers
EV: / CD81/ CD63/ CD9
non-EV: VEGF
Proteomics
no
Show all info
Study aim
Function/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
mesenchymal stem cells
EV-harvesting Medium
Serum free medium
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Ultra filtration
Cut-off size (kDa)
3
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
22
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
ELISA
Detected EV-associated proteins
Not detected EV-associated proteins
Not detected contaminants
CD63/ CD81/ CD9
Other 1
Dot Blot
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
125
EV concentration
Yes
EV190075 3/3 Homo sapiens Cell culture supernatant Filtration
SEC
Size-exclusion chromatography (non-commercial)
UF
Thomas E. Whittaker 2020 33%

Study summary

Full title
All authors
Thomas E. Whittaker, Anika Nagelkerke , Valeria Nele , Ulrike Kauscher & Molly M. Stevens
Journal
J Extracell Vesicles
Abstract
It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can (show more...)It has been demonstrated that some commonly used Extracellular Vesicle (EV) isolation techniques can lead to substantial contamination with non-EV factors. Whilst it has been established that this impacts the identification of biomarkers, the impact on apparent EV bioactivity has not been explored. Extracellular vesicles have been implicated as critical mediators of therapeutic human mesenchymal stem cell (hMSC) paracrine signalling. Isolated hMSC-EVs have been used to treat multiple in vitro and in vivo models of tissue damage. However, the relative contributions of EVs and non-EV factors have not been directly compared. The dependence of hMSC paracrine signalling on EVs was first established by ultrafiltration of hMSC-conditioned medium to deplete EVs, which led to a loss of signalling activity. Here, we show that this method also causes depletion of non-EV factors, and that when this is prevented proangiogenic signalling activity is fully restored in vitro. Subsequently, we used size-exclusion chromatography (SEC) to separate EVs and soluble proteins to directly and quantitatively compare their relative contributions to signalling. Non-EV factors were found to be necessary and sufficient for the stimulation of angiogenesis and wound healing in vitro. EVs in isolation were found to be capable of potentiating signalling only when isolated by a low-purity method, or when used at comparatively high concentrations. These results indicate a potential for contaminating soluble factors to artefactually increase the apparent bioactivity of EV isolates and could have implications for future studies on the biological roles of EVs. (hide)
EV-METRIC
33% (65th 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
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
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
Filtration + SEC + Size-exclusion chromatography (non-commercial) + UF
Protein markers
EV: / CD81/ CD63/ CD9
non-EV: VEGF
Proteomics
no
Show all info
Study aim
Function/New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
mesenchymal stem cells
EV-harvesting Medium
Serum free medium
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Ultra filtration
Cut-off size (kDa)
3
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
22
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
ELISA
Detected EV-associated proteins
Not detected EV-associated proteins
Not detected contaminants
CD63/ CD81/ CD9
Other 1
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
116
EV concentration
Yes
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