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You searched for: EV190077 (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
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV190077 1/4 Bos taurus Milk DG
(d)(U)C
milk acidification
casein removal
SEC
Andrea Ridolfi 2020 75%

Study summary

Full title
All authors
Andrea Ridolfi, Marco Brucale, Costanza Montis, Lucrezia Caselli, Lucia Paolini, Anne Borup, Anders T Boysen, Francesca Loria, Martijn J C van Herwijnen, Marije Kleinjan, Peter Nejsum, Natasa Zarovni, Marca H M Wauben, Debora Berti, Paolo Bergese, Francesco Valle
Journal
Anal Chem
Abstract
The mechanical properties of extracellular vesicles (EVs) are known to influence their biological fu (show more...)The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role. (hide)
EV-METRIC
75% (84th 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
Milk
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
milk acidification
casein removal
SEC
Protein markers
EV: TSG101/ CD63/ Flotillin1/ CD9/ MFGE8
non-EV: beta-lactoglobulin/ Casein
Proteomics
no
EV density (g/ml)
1.06-1.19
Show all info
Study aim
Other/Biophysical characterization
Sample
Species
Bos taurus
Sample Type
Milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
16
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12.45
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
197000
Duration (min)
960
Fraction volume (mL)
0.5
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
15
Sample volume/column (mL)
2
Resin type
Sephadex G-100
Other
Name other separation method
milk acidification
Other
Name other separation method
casein removal
Characterization: Protein analysis
Protein Concentration Method
Other;Colorimetric Nanoplasmonic Assay
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFGE8/ TSG101
Detected contaminants
beta-lactoglobulin
Not detected contaminants
Casein
Characterization: Lipid analysis
No
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
40-160
EV190077 3/4 Ascaris suum Ascaris suum supernatant (d)(U)C
UF
Andrea Ridolfi 2020 29%

Study summary

Full title
All authors
Andrea Ridolfi, Marco Brucale, Costanza Montis, Lucrezia Caselli, Lucia Paolini, Anne Borup, Anders T Boysen, Francesca Loria, Martijn J C van Herwijnen, Marije Kleinjan, Peter Nejsum, Natasa Zarovni, Marca H M Wauben, Debora Berti, Paolo Bergese, Francesco Valle
Journal
Anal Chem
Abstract
The mechanical properties of extracellular vesicles (EVs) are known to influence their biological fu (show more...)The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role. (hide)
EV-METRIC
29% (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. 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
Ascaris suum supernatant
Sample origin
Mycoplasma-contaminated
Focus vesicles
extracellular vesicle
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
UF
Protein markers
EV:
non-EV:
Proteomics
no
Show all info
Study aim
Other/Biophysical characterization
Sample
Species
Ascaris suum
Sample Type
Ascaris suum supernatant
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
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)
70
Pelleting: rotor type
Type 50
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
9
Wash: time (min)
70
Wash: Rotor Type
Type 50
Wash: speed (g)
110000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Protein Concentration Method
Other;Colorimetric Nanoplasmonic Assay
Other 1
There are no specific Ascaris suum EV protein markers identified yet;Kan je dit hier verwijderen en in comments plaatsen? En comments ook toevoegen aan de print?
Detected EV-associated proteins
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
166
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
60-240
EV190077 2/4 Ascaris suum Ascaris suum supernatant (d)(U)C
qEV
UF
Andrea Ridolfi 2020 17%

Study summary

Full title
All authors
Andrea Ridolfi, Marco Brucale, Costanza Montis, Lucrezia Caselli, Lucia Paolini, Anne Borup, Anders T Boysen, Francesca Loria, Martijn J C van Herwijnen, Marije Kleinjan, Peter Nejsum, Natasa Zarovni, Marca H M Wauben, Debora Berti, Paolo Bergese, Francesco Valle
Journal
Anal Chem
Abstract
The mechanical properties of extracellular vesicles (EVs) are known to influence their biological fu (show more...)The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role. (hide)
EV-METRIC
17% (33rd 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
Ascaris suum 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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
qEV
UF
Protein markers
EV:
non-EV:
Proteomics
no
Show all info
Study aim
Other/Biophysical characterization
Sample
Species
Ascaris suum
Sample Type
Ascaris suum supernatant
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
None
Protein Concentration Method
Other;Colorimetric Nanoplasmonic Assay
Other 1
There are no specific Ascaris suum EV protein markers identified yet;Kan je dit hier verwijderen en in comments plaatsen? En comments ook toevoegen aan de print?
Detected EV-associated proteins
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
165
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
60-240
EV190077 4/4 Ascaris suum Ascaris suum supernatant (d)(U)C
qEV
UF
Andrea Ridolfi 2020 17%

Study summary

Full title
All authors
Andrea Ridolfi, Marco Brucale, Costanza Montis, Lucrezia Caselli, Lucia Paolini, Anne Borup, Anders T Boysen, Francesca Loria, Martijn J C van Herwijnen, Marije Kleinjan, Peter Nejsum, Natasa Zarovni, Marca H M Wauben, Debora Berti, Paolo Bergese, Francesco Valle
Journal
Anal Chem
Abstract
The mechanical properties of extracellular vesicles (EVs) are known to influence their biological fu (show more...)The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role. (hide)
EV-METRIC
17% (33rd 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
Ascaris suum supernatant
Sample origin
Mycoplasma-contaminated
Focus vesicles
extracellular vesicle
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
qEV
UF
Protein markers
EV:
non-EV:
Proteomics
no
Show all info
Study aim
Other/Biophysical characterization
Sample
Species
Ascaris suum
Sample Type
Ascaris suum supernatant
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Commercial kit
qEV
Other
Name other separation method
qEV
Protein Concentration Method
Other;Colorimetric Nanoplasmonic Assay
Other 1
There are no specific Ascaris suum EV protein markers identified yet
Detected EV-associated proteins
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
157
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
60-240
1 - 4 of 4
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV190077
species
Bos taurus
Ascaris suum
Ascaris suum
Ascaris suum
sample type
Milk
Ascaris
suum supernatant
Ascaris
suum supernatant
Ascaris
suum supernatant
condition
Control condition
Mycoplasma-contaminated
Control condition
Mycoplasma-contaminated
separation protocol
DG
(d)(U)C
milk acidification
casein removal
SEC
(d)(U)C
UF
(d)(U)C
qEV
UF
(d)(U)C
qEV
UF
Exp. nr.
1
3
2
4
EV-METRIC %
75
29
17
17