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You searched for: EV200102 (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 code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV200102 1/7 Homo sapiens Cell culture supernatant DG
(d)(U)C
qEV
Tóth, Eszter 2021 100%

Study summary

Full title
All authors
Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás
Journal
J Extracell Vesicles
Abstract
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (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. 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
Density gradient
(Differential) (ultra)centrifugation
Commercial method
Protein markers
EV: CD63/ Phosphatydilserine
non-EV: None
Proteomics
yes
EV density (g/ml)
1.10-1.15
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
THP1
EV-harvesting Medium
Serum free medium
Cell viability (%)
93
Cell count
80000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting: time(min)
40
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
12500
Wash: volume per pellet (ml)
1
Wash: time (min)
40
Wash: Rotor Type
FA-45-24-11
Wash: speed (g)
12500
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
2
Pelleting: duration (min)
80
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
12500
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Type of Flow cytometry
FACS Calibur
Calibration bead size
The vesicular gate was set using Megamix Beads (Bi
Antibody details provided?
No
Detected EV-associated proteins
Phosphatydilserine
Proteomics database
Yes:
Detected EV-associated proteins
CD63
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
244
Particle analysis: flow cytometry
Flow cytometer type
FACS Calibur
Hardware adjustment
Calibration bead size
0.160;0.200;0.240;0.500
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200102 4/7 Homo sapiens Blood plasma DG Tóth, Eszter 2021 86%

Study summary

Full title
All authors
Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás
Journal
J Extracell Vesicles
Abstract
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide)
EV-METRIC
86% (98th 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
Blood plasma
Sample origin
Control: EV-depleted plasma, spiked with THP1 EVs
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
Density gradient
Protein markers
EV: CD63/ Phosphatydilserine
non-EV: None
Proteomics
yes
EV density (g/ml)
1.10-1.15
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
2
Pelleting: duration (min)
80
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
12500
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Type of Flow cytometry
FACS Calibur
Calibration bead size
The vesicular gate was set using Megamix Beads (Bi
Antibody details provided?
No
Detected EV-associated proteins
Phosphatydilserine
Proteomics database
Yes:
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
FACS Calibur
Hardware adjustment
Calibration bead size
0.160;0.200;0.240;0.500
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200102 2/7 Homo sapiens Blood plasma (d)(U)C Tóth, Eszter 2021 75%

Study summary

Full title
All authors
Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás
Journal
J Extracell Vesicles
Abstract
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide)
EV-METRIC
75% (97th 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
Blood plasma
Sample origin
Control: EV-depleted plasma, spiked with THP1 EVs
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ Phosphatydilserine
non-EV: Albumin/ fibrinogen/ haptoglobin/ complement C3
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting: time(min)
40
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
12500
Wash: volume per pellet (ml)
1
Wash: time (min)
40
Wash: Rotor Type
FA-45-24-11
Wash: speed (g)
12500
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Type of Flow cytometry
FACS Calibur
Calibration bead size
The vesicular gate was set using Megamix Beads (Bi
Antibody details provided?
No
Detected EV-associated proteins
Phosphatydilserine
Proteomics database
Yes:
Detected EV-associated proteins
CD63
Detected contaminants
fibrinogen/ haptoglobin/ complement C3/ Albumin
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
240
Particle analysis: flow cytometry
Flow cytometer type
FACS Calibur
Hardware adjustment
Calibration bead size
0.160;0.200;0.240;0.500
Report type
Not Reported
EM
EM-type
Immuno-EM/ Transmission-EM
Proteïns
Other;CD63, plasma proteins
Image type
Close-up, Wide-field
EV200102 5/7 Homo sapiens Blood plasma (d)(U)C Tóth, Eszter 2021 75%

Study summary

Full title
All authors
Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás
Journal
J Extracell Vesicles
Abstract
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide)
EV-METRIC
75% (97th 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
Blood plasma
Sample origin
Rheuma: EV-depleted plasma, spiked with THP1 EVs
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
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ Phosphatydilserine
non-EV: Albumin/ fibrinogen/ haptoglobin/ complement C3
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting: time(min)
40
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
12500
Wash: volume per pellet (ml)
1
Wash: time (min)
40
Wash: Rotor Type
FA-45-24-11
Wash: speed (g)
12500
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Type of Flow cytometry
FACS Calibur
Calibration bead size
The vesicular gate was set using Megamix Beads (Bi
Antibody details provided?
No
Detected EV-associated proteins
Phosphatydilserine
Proteomics database
Yes:
Detected EV-associated proteins
CD63
Detected contaminants
fibrinogen/ haptoglobin/ complement C3/ Albumin
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Not Reported
Particle analysis: flow cytometry
Flow cytometer type
FACS Calibur
Hardware adjustment
Calibration bead size
0.160;0.200;0.240;0.500
Report type
Not Reported
EV200102 7/7 Homo sapiens Blood plasma DG Tóth, Eszter 2021 71%

Study summary

Full title
All authors
Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás
Journal
J Extracell Vesicles
Abstract
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide)
EV-METRIC
71% (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. 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
Blood plasma
Sample origin
Rheuma: EV-depleted plasma, spiked with THP1 EVs
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
Density gradient
Protein markers
EV: CD63/ Phosphatydilserine
non-EV: None
Proteomics
yes
EV density (g/ml)
1.10-1.15
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
4.5
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
2
Pelleting: duration (min)
80
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
12500
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Type of Flow cytometry
FACS Calibur
Calibration bead size
The vesicular gate was set using Megamix Beads (Bi
Antibody details provided?
No
Detected EV-associated proteins
Phosphatydilserine
Proteomics database
Yes:
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
FACS Calibur
Hardware adjustment
Calibration bead size
0.160;0.200;0.240;0.500
Report type
Not Reported
EV200102 3/7 Homo sapiens Blood plasma qEV Tóth, Eszter 2021 57%

Study summary

Full title
All authors
Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás
Journal
J Extracell Vesicles
Abstract
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide)
EV-METRIC
57% (92nd 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
Blood plasma
Sample origin
Control: EV-depleted plasma, spiked with THP1 EVs
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
qEV
Protein markers
EV: CD63/ Phosphatydilserine
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Type of Flow cytometry
FACS Calibur
Calibration bead size
The vesicular gate was set using Megamix Beads (Bi
Antibody details provided?
No
Detected EV-associated proteins
Phosphatydilserine
Proteomics database
Yes:
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
FACS Calibur
Hardware adjustment
Calibration bead size
0.160;0.200;0.240;0.500
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200102 6/7 Homo sapiens Blood plasma qEV Tóth, Eszter 2021 43%

Study summary

Full title
All authors
Eszter Á Tóth, Lilla Turiák, Tamás Visnovitz, Csaba Cserép, Anett Mázló, Barbara W Sódar, András I Försönits, Gábor Petővári, Anna Sebestyén, Zsolt Komlósi, László Drahos, Ágnes Kittel, György Nagy, Attila Bácsi, Ádám Dénes, Yong Song Gho, Katalin É Szabó-Taylor, Edit I Buzás
Journal
J Extracell Vesicles
Abstract
In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in bl (show more...)In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research. (hide)
EV-METRIC
43% (77th 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
Blood plasma
Sample origin
Rheuma: EV-depleted plasma, spiked with THP1 EVs
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
qEV
Protein markers
EV: CD63/ Phosphatydilserine
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Flow cytometry
Type of Flow cytometry
FACS Calibur
Calibration bead size
The vesicular gate was set using Megamix Beads (Bi
Antibody details provided?
No
Detected EV-associated proteins
Phosphatydilserine
Proteomics database
Yes:
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
FACS Calibur
Hardware adjustment
Calibration bead size
0.160;0.200;0.240;0.500
Report type
Not Reported
1 - 7 of 7
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200102
species
Homo
sapiens
sample type
Cell
culture
Blood
plasma
Blood
plasma
Blood
plasma
Blood
plasma
Blood
plasma
Blood
plasma
cell type
THP1
NA
NA
NA
NA
NA
NA
medium
Serum
free
medium
NA
NA
NA
NA
NA
NA
condition
Control
condition
Control: EV-depleted plasma
spiked
with
THP1
EVs
Control: EV-depleted plasma
spiked
with
THP1
EVs
Rheuma: EV-depleted plasma
spiked
with
THP1
EVs
Rheuma: EV-depleted plasma
spiked
with
THP1
EVs
Control: EV-depleted plasma
spiked
with
THP1
EVs
Rheuma: EV-depleted plasma
spiked
with
THP1
EVs
separation protocol
Density
gradient
dUC
qEV
Density
gradient
dUC
dUC
Density
gradient
qEV
qEV
Exp. nr.
1
4
2
5
7
3
6
EV-METRIC %
100
86
75
75
71
57
43