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Results list Study Tree
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
EV200001 1/9 Homo sapiens EBC1 DG
(d)(U)C 		Useckaite, Zivile 2020 89%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
89% (99th 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
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
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9
non-EV: GRP94
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
EBC1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
99
Cell count
2.25E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Density gradient
Type
Continuous
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
1080
Fraction volume (mL)
5
Fraction processing
Centrifugation
Pelleting: volume per fraction
17;17mL
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32.1 Ti;SW 32 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Syntenin1/ Actinin4
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
111
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 86300000000
EM
EM-type
Transmission-EM
Image type
Close-up
EV200001 4/9 Homo sapiens H596 DG
(d)(U)C 		Useckaite, Zivile 2020 89%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
89% (99th 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
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
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9
non-EV: GRP94
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
H596
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
98
Cell count
2.03E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Density gradient
Type
Continuous
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
1080
Fraction volume (mL)
5
Fraction processing
Centrifugation
Pelleting: volume per fraction
17;17mL
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32.1 Ti;SW 32 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Syntenin1/ Actinin4/ CD9/ CD63
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
106
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 89500000000
EM
EM-type
Transmission-EM
Image type
Close-up
EV200001 7/9 Homo sapiens Hs746T DG
(d)(U)C 		Useckaite, Zivile 2020 89%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
89% (99th 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
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
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9
non-EV: GRP94
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Hs746T
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
98
Cell count
2.03E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Density gradient
Type
Continuous
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
1080
Fraction volume (mL)
5
Fraction processing
Centrifugation
Pelleting: volume per fraction
17;17mL
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32.1 Ti;SW 32 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Syntenin1/ Actinin4/ CD9/ CD63
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
107
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 29000000000
EM
EM-type
Transmission-EM
Image type
Close-up
EV200001 2/9 Homo sapiens EBC1 (d)(U)C Useckaite, Zivile 2020 67%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
67% (94th 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
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
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
EBC1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
99
Cell count
2.25E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Syntenin1/ Actinin4
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
158
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 20800000000
EM
EM-type
Transmission-EM
Image type
Close-up
EV200001 3/9 Homo sapiens EBC1 (d)(U)C
ExoQuick 		Useckaite, Zivile 2020 67%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
67% (94th 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
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
ExoQuick
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ GRP94/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
EBC1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
99
Cell count
2.25E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Other
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Syntenin1/ GRP94
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
119
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 771000000
EM
EM-type
Transmission-EM
Image type
Close-up
EV200001 5/9 Homo sapiens H596 (d)(U)C Useckaite, Zivile 2020 67%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
67% (94th 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
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
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
H596
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
98
Cell count
2.03E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Syntenin1/ Actinin4/ CD9/ CD63
Detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
146
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 11400000000
EM
EM-type
Transmission-EM
Image type
Close-up
EV200001 6/9 Homo sapiens H596 (d)(U)C
ExoQuick 		Useckaite, Zivile 2020 67%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
67% (94th 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
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
ExoQuick
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
H596
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
98
Cell count
2.03E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Syntenin1/ Actinin4/ CD9/ CD63
Detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
123
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 5710000000
EM
EM-type
Transmission-EM
Image type
Close-up
EV200001 8/9 Homo sapiens Hs746T (d)(U)C Useckaite, Zivile 2020 67%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
67% (94th 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
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
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ Actinin4/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Hs746T
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
98
Cell count
2.03E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Syntenin1/ Actinin4/ CD9/ CD63
Detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
147
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 11600000000
EM
EM-type
Transmission-EM
Image type
Close-up
EV200001 9/9 Homo sapiens Hs746T (d)(U)C
ExoQuick 		Useckaite, Zivile 2020 67%

Study summary

Full title
Extracellular vesicles report on the MET status of their cells of origin regardless of the method used for their isolation
All authors
Zivile Useckaite, Anindya Mukhopadhya, Barry Moran, Lorraine O'Driscoll
Journal
Sci Rep
Abstract
MET pathway is an important actionable target across many solid tumour types and several MET inhibit (show more...)MET pathway is an important actionable target across many solid tumour types and several MET inhibitors have been developed. Extracellular vesicles (EVs) are proposed to be mini-maps of their cells of origin. However, the potential of EVs to report on the MET status of their cells of origin is unknown. After applying three proposed methods of EV separation from medium conditioned by three cell lines of known MET status, this study used an extensive range of methodologies to fundamentally characterise the resulting particles (nanoparticle tracking analysis, TEM, flow cytometry, immunoblotting) and their MET status (RT-qPCR and ELISAs). The results indicated that ultracentrifugation on density-gradient (UC-DG) consistently produced the most reliable data with regards to purest EVs. EV cargo reflected MET mRNA, total MET and pMET status of their cells of origin. In conclusion, to simply determine if the general contents of conditioned medium reflect the MET status of the conditioning cells, choice of method for initial EV separation may not be crucial. However, to be confident of specifically studying EVs and thus EV-MET cargo, UC-DG followed by extensive EV characterisation is necessary. (hide)
EV-METRIC
67% (94th 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
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
ExoQuick
Protein markers
EV: Syntenin1/ CD63/ CD81/ HLA-DR/ pMET (pY1234/1235)/ ADAM10/ MET/ GRP94/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Hs746T
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
18h, 120000g;Other preparation
Cell viability (%)
98
Cell count
2.03E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
10000
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Syntenin1/ GRP94/ CD9/ CD63
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
MET/ pMET (pY1234/1235)
Flow cytometry
Type of Flow cytometry
AMNIS ImageStreamX Mark II
Hardware adaptation to ~100nm EV's
Laser powers were adjusted to ensure the fluorophore intensity was within the detection range. Fluorescent signals were collected using the following channels: FITC was measured in channel 2 (480560 n
Calibration bead size
80+
Detected EV-associated proteins
CD63/ CD9/ CD81/ ADAM10/ HLA-DR
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
111
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 3740000000
EM
EM-type
Transmission-EM
Image type
Close-up
1 - 9 of 9
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200001
species
Homo
sapiens
sample type
Cell
culture
cell type
EBC1
H596
Hs746T
EBC1
EBC1
H596
H596
Hs746T
Hs746T
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
separation protocol
DG
(d)(U)C
DG
(d)(U)C
DG
(d)(U)C
(d)(U)C
(d)(U)C
ExoQuick
(d)(U)C
(d)(U)C
ExoQuick
(d)(U)C
(d)(U)C
ExoQuick
Exp. nr.
1
4
7
2
3
5
6
8
9
EV-METRIC %
89
89
89
67
67
67
67
67
67