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You searched for: EV190018 (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
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV190018 3/8 Homo sapiens Cell culture supernatant DG
dUC
Filtration
(Differential) (ultra)centrifugation
Filtration
Density gradient
Freitas D 2019 100%

Study summary

Full title
All authors
Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide)
EV-METRIC
100% (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
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
Sample origin
Control condition
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 + (Differential) (ultra)centrifugation + Filtration + Density gradient
Protein markers
EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1
non-EV: Albumin
Proteomics
yes
EV density (g/ml)
1.05-1.10
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MKN45
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
5.5
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Ultrafiltration
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Other;silver staining
Western Blot
Detected EV-associated proteins
Alix/ CD9/ CD63/ HSP70/ Syntenin-1
Not detected EV-associated proteins
CD81
Detected contaminants
Albumin
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
131.65
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190018 7/8 Homo sapiens Cell culture supernatant DG
dUC
Filtration
(Differential) (ultra)centrifugation
Filtration
Density gradient
Freitas D 2019 100%

Study summary

Full title
All authors
Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide)
EV-METRIC
100% (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
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
Sample origin
COSMC KO
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 + (Differential) (ultra)centrifugation + Filtration + Density gradient
Protein markers
EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1
non-EV: Albumin
Proteomics
yes
EV density (g/ml)
1.05-1.10
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
COSMC KO
EV-producing cells
MKN45
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
5.5
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Ultrafiltration
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Other;silver staining
Western Blot
Detected EV-associated proteins
Alix/ Syntenin-1/ CD9/ CD63/ HSP70/ CD81
Detected contaminants
Albumin
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
121.05
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190018 1/8 Homo sapiens Cell culture supernatant dUC
Filtration
(Differential) (ultra)centrifugation
Filtration
Freitas D 2019 75%

Study summary

Full title
All authors
Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide)
EV-METRIC
75% (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
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + (Differential) (ultra)centrifugation + Filtration
Protein markers
EV: Alix/ CD63/ CD81/ Proteins regarded in the experiment as potentially EV-associated and not detected in the EVs/ HSP70/ CD9/ Syntenin-1
non-EV: Cytochrome C/ Albumin
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MKN45
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
960
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38
Wash: time (min)
120
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Other;silver staining
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Syntenin-1/ HSP70/ Alix/ CD81
Not detected EV-associated proteins
Proteins regarded in the experiment as potentially EV-associated and not detected in the EVs
Detected contaminants
Albumin
Not detected contaminants
Cytochrome C
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
140.97
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190018 2/8 Homo sapiens Cell culture supernatant Total Exosome Isolation
Filtration
dUC
Freitas D 2019 75%

Study summary

Full title
All authors
Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide)
EV-METRIC
75% (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
Sample origin
Control condition
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
Total Exosome Isolation + Filtration + dUC
Protein markers
EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1
non-EV: Cytochrome C/ Albumin
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MKN45
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
Other;silver staining
Western Blot
Detected EV-associated proteins
CD63/ Syntenin-1/ HSP70/ CD81
Not detected EV-associated proteins
CD9/ Alix
Detected contaminants
Albumin
Not detected contaminants
Cytochrome C
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
148.3
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190018 4/8 Homo sapiens Cell culture supernatant dUC
Filtration
qEV
Freitas D 2019 75%

Study summary

Full title
All authors
Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide)
EV-METRIC
75% (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
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + qEV
Protein markers
EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MKN45
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Other;silver staining
Western Blot
Detected EV-associated proteins
Syntenin-1/ CD9/ CD63/ HSP70/ CD81
Not detected EV-associated proteins
Alix
Detected contaminants
Albumin
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
138.37
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190018 5/8 Homo sapiens Cell culture supernatant dUC
Filtration
(Differential) (ultra)centrifugation
Filtration
Freitas D 2019 75%

Study summary

Full title
All authors
Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide)
EV-METRIC
75% (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
Sample origin
COSMC KO
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + (Differential) (ultra)centrifugation + Filtration
Protein markers
EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
COSMC KO
EV-producing cells
MKN45
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
960
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38
Wash: time (min)
120
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Other;silver staining
Western Blot
Detected EV-associated proteins
Syntenin-1/ CD9/ CD63/ HSP70/ Alix/ CD81
Detected contaminants
Albumin
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
141.43
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190018 6/8 Homo sapiens Cell culture supernatant Total Exosome Isolation
Filtration
dUC
Freitas D 2019 75%

Study summary

Full title
All authors
Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide)
EV-METRIC
75% (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
Sample origin
COSMC KO
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
Total Exosome Isolation + Filtration + dUC
Protein markers
EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
COSMC KO
EV-producing cells
MKN45
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
Other;silver staining
Western Blot
Detected EV-associated proteins
Alix/ Syntenin-1/ CD63/ HSP70/ CD81
Not detected EV-associated proteins
CD9
Detected contaminants
Albumin
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
152.5
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190018 8/8 Homo sapiens Cell culture supernatant dUC
Filtration
qEV
Freitas D 2019 75%

Study summary

Full title
All authors
Freitas D, Balmaña M, Poças J, Campos D, Osório H, Konstantinidi A, Vakhrushev SY, Magalhães A, Reis CA.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in inter (show more...)Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates. (hide)
EV-METRIC
75% (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
Sample origin
COSMC KO
Focus vesicles
extracellular vesicle
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration + qEV
Protein markers
EV: CD63/ CD81/ Alix/ HSP70/ CD9/ Syntenin-1
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
COSMC KO
EV-producing cells
MKN45
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Other;silver staining
Western Blot
Detected EV-associated proteins
Alix/ Syntenin-1/ CD9/ CD63/ HSP70/ CD81
Detected contaminants
Albumin
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
148.33
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
1 - 8 of 8
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV190018
species
Homo
sapiens
sample type
Cell
culture
cell type
MKN45
condition
Control
condition
COSMC
KO
Control
condition
Control
condition
Control
condition
COSMC
KO
COSMC
KO
COSMC
KO
isolation protocol
DG
dUC
Filtration
dUC
Filtration
Density
gradient
DG
dUC
Filtration
dUC
Filtration
Density
gradient
dUC
Filtration
dUC
Filtration
Total
Exosome
Isolation
Filtration
dUC
dUC
Filtration
qEV
dUC
Filtration
dUC
Filtration
Total
Exosome
Isolation
Filtration
dUC
dUC
Filtration
qEV
Exp. nr.
3
7
1
2
4
5
6
8
EV-METRIC %
100
100
75
75
75
75
75
75