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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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV190064 5/10 Homo sapiens Urine DG
dUC
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
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
Urine
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
DG + dUC + UF
Protein markers
EV: TSG101/ Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 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)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101/ Alix
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
196.5
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 6/10 Homo sapiens Urine DG
dUC
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
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
Urine
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
DG + dUC + UF
Protein markers
EV: TSG101/ Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 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)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101/ Alix
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
131.7
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 7/10 Homo sapiens Urine DG
dUC
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
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
Urine
Sample origin
Prostate Cancer
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
DG + dUC + UF
Protein markers
EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1
non-EV: Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Prostate Cancer
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 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)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9
Detected contaminants
Tamm-Horsfall protein
Proteomics database
Yes:
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 8/10 Homo sapiens Urine DG
dUC
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
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
Urine
Sample origin
Bladder Cancer
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
DG + dUC + UF
Protein markers
EV: Alix/ Flotillin1/ CD9/ Syntenin-1
non-EV: Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Bladder Cancer
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 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)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Syntenin-1/ CD9
Detected contaminants
Tamm-Horsfall protein
Proteomics database
Yes:
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 9/10 Homo sapiens Urine DG
dUC
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
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
Urine
Sample origin
Renal Cancer
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
DG + dUC + UF
Protein markers
EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1
non-EV: Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Renal Cancer
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 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)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9
Detected contaminants
Tamm-Horsfall protein
Proteomics database
Yes:
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
100-300
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 10/10 Homo sapiens Tissue DG
dUC
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Tissue
Sample origin
Prostate Cancer
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
DG + dUC + UF
Protein markers
EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1
non-EV: Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Tissue
Sample Condition
Prostate Cancer
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 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)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9
Detected contaminants
Tamm-Horsfall protein
Proteomics database
Yes
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
150.3
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190084 1/2 Homo sapiens Cell culture supernatant dUC
Filtration
Greet Merckx 2020 78%

Study summary

Full title
All authors
Greet Merckx, Baharak Hosseinkhani, Sören Kuypers, Sarah Deville, Joy Irobi, Inge Nelissen, Luc Michiels, Ivo Lambrichts, Annelies Bronckaers
Journal
Cells
Abstract
Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marr (show more...)Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis. (hide)
EV-METRIC
78% (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
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
dUC + Filtration
Protein markers
EV: CD9/ CD63/ ANXA2/ CD81
non-EV: Bax
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Dental pulp stromal cells
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
180
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ ANXA2/ CD81
Not detected contaminants
Bax
NA
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190084 2/2 Homo sapiens Cell culture supernatant dUC
Filtration
Greet Merckx 2020 78%

Study summary

Full title
All authors
Greet Merckx, Baharak Hosseinkhani, Sören Kuypers, Sarah Deville, Joy Irobi, Inge Nelissen, Luc Michiels, Ivo Lambrichts, Annelies Bronckaers
Journal
Cells
Abstract
Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marr (show more...)Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis. (hide)
EV-METRIC
78% (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
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
dUC + Filtration
Protein markers
EV: CD9/ CD63/ ANXA2/ CD81
non-EV: Bax
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Bone marrow derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
180
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ ANXA2/ CD81
Not detected contaminants
Bax
NA
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190079 1/2 Homo sapiens kidney tissue supernatant dUC
Filtration
Zieren RC 2020 78%

Study summary

Full title
All authors
Zieren RC, Dong L, Pierorazio PM, Pienta KJ, de Reijke TM, Amend SR.
Journal
Med Oncol
Abstract
Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive bio (show more...)Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive biomarkers are needed to aid diagnosis and treatment. Extracellular vesicles (EVs), membranous vesicles secreted by all cells, are a promising potential source for cancer biomarkers, but new methods are required that are both sensitive and specific for cancer identification. We have developed an EV isolation protocol optimized for kidney tumor and normal kidney tissue that yields a high vesicle concentration, confirmed by nanoparticle tracking analysis (NanoSight) and by nanoscale flow cytometry (NanoFCM). Using Western blot, we confirmed presence of EV markers CD81, CD63, flotillin-1, and absence of cellular debris, calnexin. Transmission electron microscopy images demonstrate intact membranous EVs. This new method improves existing protocols with additional steps to reduce contaminants in the EV product. Characterization of our isolation product confirms successful isolation of EVs with minimal contamination. The particle yields of our protocol are consistent and high as assessed by both standard and novel methods. This optimized protocol will contribute to biomarker discovery and biological studies of EVs in renal cancer. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
kidney tissue 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
dUC + Filtration
Protein markers
EV: CD81/ CD63/ Flotillin1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
kidney tissue supernatant
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Filtration steps
> 0.45 µm, 0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calnexin
Characterization: Particle analysis
NA
NTA
Report type
Modus
Reported size (nm)
163
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Instrument was manufactured for small EVs
Calibration bead size
200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report type
Modus
Report size
57
EV-concentration
Yes
EV190079 2/2 Homo sapiens kidney tissue supernatant dUC
Filtration
Zieren RC 2020 78%

Study summary

Full title
All authors
Zieren RC, Dong L, Pierorazio PM, Pienta KJ, de Reijke TM, Amend SR.
Journal
Med Oncol
Abstract
Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive bio (show more...)Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive biomarkers are needed to aid diagnosis and treatment. Extracellular vesicles (EVs), membranous vesicles secreted by all cells, are a promising potential source for cancer biomarkers, but new methods are required that are both sensitive and specific for cancer identification. We have developed an EV isolation protocol optimized for kidney tumor and normal kidney tissue that yields a high vesicle concentration, confirmed by nanoparticle tracking analysis (NanoSight) and by nanoscale flow cytometry (NanoFCM). Using Western blot, we confirmed presence of EV markers CD81, CD63, flotillin-1, and absence of cellular debris, calnexin. Transmission electron microscopy images demonstrate intact membranous EVs. This new method improves existing protocols with additional steps to reduce contaminants in the EV product. Characterization of our isolation product confirms successful isolation of EVs with minimal contamination. The particle yields of our protocol are consistent and high as assessed by both standard and novel methods. This optimized protocol will contribute to biomarker discovery and biological studies of EVs in renal cancer. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
kidney tissue supernatant
Sample origin
kidney cancer
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
dUC + Filtration
Protein markers
EV: CD81/ CD63/ Flotillin1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
kidney tissue supernatant
Sample Condition
kidney cancer
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Filtration steps
> 0.45 µm, 0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calnexin
Characterization: Particle analysis
NA
NTA
Report type
Modus
Reported size (nm)
133
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Instrument was manufactured for small EVs
Calibration bead size
200
Report type
Modus
Reported size (nm)
57
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report type
Modus
Report size
57
EV-concentration
Yes
EV190076 1/1 Homo sapiens Urine dUC Musante L 2020 78%

Study summary

Full title
All authors
Musante L, Bontha SV, La Salvia S, Fernandez-Piñeros A, Lannigan J, Le TH, Mas V, Erdbrügger U
Journal
Sci Rep
Abstract
Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centri (show more...)Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. Cryo-Transmission Electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of EVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single EV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. This work characterizes a neglected source of uEVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of uEV analysis such as autofluorescence of podocyte origin. (hide)
EV-METRIC
78% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
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
dUC
Protein markers
EV: TSG101/ Podocin/ Podocalyxin/ Collectrin/ IGFBP7/ CD9
non-EV: Calnexin/ Tamm-Horsfall protein/ Albumin/ Calreticulin
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
30
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
21130
Wash: volume per pellet (ml)
1.2
Wash: time (min)
30
Wash: Rotor Type
FA-45-24-11
Wash: speed (g)
21130
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD9/ Podocalyxin/ Collectrin/ Podocin/ TSG101
Detected contaminants
Calnexin/ Calreticulin/ Albumin/ Tamm-Horsfall protein
Flow cytometry
Type of Flow cytometry
ImageStreamX Mark II
Hardware adjustments
Imaging flow cytometry (IFCM) is a method combining flow cytometry with imaging. All signals are collected through microscope objectives and quantified based on images detected by charge coupled devic
Detected EV-associated proteins
Podocalyxin/ Collectrin/ IGFBP7
Proteomics database
No
Characterization: Particle analysis
NA
NTA
Report type
Modus
Reported size (nm)
175
EV concentration
Yes
TRPS
Report type
Modus
Reported size (nm)
173
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV190064 1/10 Homo sapiens Cell culture supernatant DG
dUC
Filtration
UF
Dhondt B 2020 78%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
78% (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
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
pMET7-gag-EGFP transfected
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
DG + dUC + Filtration + UF
Protein markers
EV: Flotillin1/ Syntenin-1/ gag-EGFP
non-EV:
Proteomics
no
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
pMET7-gag-EGFP transfected
EV-producing cells
HEK293T
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
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)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ Syntenin-1/ gag-EGFP
Fluorescent NTA
Relevant measurements variables specified?
NA
Detected EV-associated proteins
gag-EGFP
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Immuno-EM
Proteïns
CD63
Image type
Close-up, Wide-field
EV190064 3/10 Homo sapiens Urine dUC
SEC
Size-exclusion chromatography (non-commercial)
UF
Dhondt B 2020 75%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
75% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
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
dUC + SEC + Size-exclusion chromatography (non-commercial) + UF
Protein markers
EV: Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ CD9
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
130
EV190089 1/1 Homo sapiens Cell culture supernatant dUC Guowen Hu 2020 56%

Study summary

Full title
All authors
Guowen Hu, Yuguo Xia, Juntao Zhang, Yu Chen, Ji Yuan, Xin Niu, Bizeng Zhao, Qing Li, Yang Wang, Zhifeng Deng
Journal
Advanced Science
Abstract
Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism (show more...)Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism is unclear and there is still lack of effective medications. In this study, the VD rats exhibit a progressive cognitive impairment, as well as a time‐related increasing in hippocampal neural stem cells (H‐NSCs) senescence, lost and neurogenesis decline. Then, embryonic stem cell‐derived small extracellular vesicles (ESC‐sEVs) are intravenously injected into VD rats. ESC‐sEVs treatment significantly alleviates H‐NSCs senescence, recovers compromised proliferation and neuron differentiation capacity, and reverses cognitive impairment. By microarray analysis and RT‐qPCR it is identified that several miRNAs including miR‐17‐5p, miR‐18a‐5p, miR‐21‐5p, miR‐29a‐3p, and let‐7a‐5p, that can inhibit mTORC1 activation, exist in ESC‐sEVs. ESC‐sEVs rejuvenate H‐NSCs senescence partly by transferring these miRNAs to inhibit mTORC1 activation, promote transcription factor EB (TFEB) nuclear translocation and lysosome resumption. Taken together, these data indicate that H‐NSCs senescence cause cell depletion, neurogenesis reduction, and cognitive impairment in VD. ESC‐sEVs treatment ameliorates H‐NSCs senescence by inhibiting mTORC1 activation, and promoting TFEB nuclear translocation and lysosome resumption, thereby reversing senescence‐related neurogenesis dysfunction and cognitive impairment in VD. The application of ESC‐sEVs may be a novel cell‐free therapeutic tool for patients with VD, as well as other aging‐related diseases. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Embryonic stem cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
114
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38.5
Wash: time (min)
114
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Not detected contaminants
GM130
NA
EV190064 2/10 Homo sapiens Urine dUC
Filtration
Dhondt B 2020 56%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
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
dUC + Filtration
Protein markers
EV: Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
16
Wash: time (min)
70
Wash: Rotor Type
SW 32.1 Ti
Wash: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ CD9
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
226.2
EV concentration
Yes
EV200068 1/5 Homo sapiens Blood plasma (Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Filtration
Linda Hofmann 2020 50%

Study summary

Full title
All authors
Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki
Journal
Int J Mol Sci
Abstract
Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide)
EV-METRIC
50% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
exosome
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
(Differential) (ultra)centrifugation + Size-exclusion chromatography (non-commercial) + Filtration
Protein markers
EV: TSG101/ CD81/ CD63/ CD9/ CD16
non-EV: Grp94/ ApoA1
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ CD81
Not detected contaminants
ApoA1/ Grp94
Flow cytometry aspecific beads
Detected EV-associated proteins
CD16
Flow cytometry specific beads
Selected surface protein(s)
CD44v3
Detected EV-associated proteins
CD16
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
30-150
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200068 2/5 Homo sapiens Blood plasma (Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Filtration
Linda Hofmann 2020 50%

Study summary

Full title
All authors
Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki
Journal
Int J Mol Sci
Abstract
Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide)
EV-METRIC
50% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
HNSCC
Focus vesicles
exosome
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
(Differential) (ultra)centrifugation + Size-exclusion chromatography (non-commercial) + Filtration
Protein markers
EV: TSG101/ CD81/ CD63/ CD9/ CD16
non-EV: Grp94/ ApoA1
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
HNSCC
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ CD81
Not detected contaminants
ApoA1/ Grp94
Flow cytometry aspecific beads
Detected EV-associated proteins
CD16
Flow cytometry specific beads
Selected surface protein(s)
CD44v3
Detected EV-associated proteins
CD16
NA
EV190064 4/10 Homo sapiens Urine dUC
ExoQuick
UF
Dhondt B 2020 50%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
50% (86th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
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
dUC + ExoQuick + UF
Protein markers
EV: Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Alix/ CD9
Not detected EV-associated proteins
Flotillin1
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
185.4
EV concentration
Yes
EV200032 1/11 Homo sapiens Cell culture supernatant Filtration
Commercial method
Stephanie Jung 2020 34%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
34% (71st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control
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
Filtration + Commercial method
Protein markers
EV: CD63/ Syntenin/ TNF alpha
non-EV: GAPDH/ Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control
EV-producing cells
Huh7-NTC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Syntenin
Not detected contaminants
GAPDH/ Calnexin
ELISA
Detected EV-associated proteins
TNF alpha
NA
EV200032 5/11 Homo sapiens Cell culture supernatant Filtration
Commercial method
Stephanie Jung 2020 23%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
23% (54th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control
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
Filtration + Commercial method
Protein markers
EV: TNF alpha/ IFN gamma/ IL 6
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control
EV-producing cells
Hep G2-NTC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
Characterization: Protein analysis
ELISA
Detected EV-associated proteins
TNF alpha/ IFN gamma/ IL 6
Characterization: Particle analysis
NA
EM
EM-type
Transmission­-EM
Image type
Close-up, Wide-field
EV200068 3/5 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Filtration
Ultrafiltration
Linda Hofmann 2020 15%

Study summary

Full title
All authors
Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki
Journal
Int J Mol Sci
Abstract
Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide)
EV-METRIC
15% (42nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
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
(Differential) (ultra)centrifugation + Size-exclusion chromatography (non-commercial) + Filtration + Ultrafiltration
Protein markers
EV: CD16
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
THP-1
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
Commercial EV-depleted serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Flow cytometry aspecific beads
Detected EV-associated proteins
CD16
NA
EV200068 4/5 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Filtration
Ultrafiltration
Linda Hofmann 2020 15%

Study summary

Full title
All authors
Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki
Journal
Int J Mol Sci
Abstract
Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide)
EV-METRIC
15% (42nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
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
(Differential) (ultra)centrifugation + Size-exclusion chromatography (non-commercial) + Filtration + Ultrafiltration
Protein markers
EV: CD16
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PCI-30
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
Commercial EV-depleted serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Flow cytometry aspecific beads
Detected EV-associated proteins
CD16
NA
EV200068 5/5 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Filtration
Ultrafiltration
Linda Hofmann 2020 15%

Study summary

Full title
All authors
Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki
Journal
Int J Mol Sci
Abstract
Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide)
EV-METRIC
15% (42nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
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
(Differential) (ultra)centrifugation + Size-exclusion chromatography (non-commercial) + Filtration + Ultrafiltration
Protein markers
EV: CD16
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
SCC-47
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
Commercial EV-depleted serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Flow cytometry aspecific beads
Detected EV-associated proteins
CD16
NA
EV200032 6/11 Homo sapiens Cell culture supernatant Filtration
Commercial method
Stephanie Jung 2020 12%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
12% (31st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
HDV infected
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
Filtration + Commercial method
Protein markers
EV: TNF alpha/ IFN gamma/ IL 6
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
HDV infected
EV-producing cells
Hep G2-NTC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
Characterization: Protein analysis
ELISA
Detected EV-associated proteins
TNF alpha/ IFN gamma/ IL 6
NA
EV200032 7/11 Homo sapiens Cell culture supernatant Filtration
Commercial method
Stephanie Jung 2020 12%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
12% (31st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
UV irradiated HDV infected
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
Filtration + Commercial method
Protein markers
EV: TNF alpha/ IFN gamma/ IL 6
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
UV irradiated HDV infected
EV-producing cells
Hep G2-NTC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
Characterization: Protein analysis
ELISA
Not detected EV-associated proteins
Not detected contaminants
TNF alpha/ IFN gamma/ IL 6
NA
EV200032 8/11 Homo sapiens Cell culture supernatant Filtration
Commercial method
Stephanie Jung 2020 12%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
12% (31st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
IFN beta treated
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
Filtration + Commercial method
Protein markers
EV: TNF alpha/ IFN gamma/ IL 6
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
IFN beta treated
EV-producing cells
Hep G2-NTC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
Characterization: Protein analysis
ELISA
Not detected EV-associated proteins
Not detected contaminants
TNF alpha/ IFN gamma/ IL 6
NA
EV200032 2/11 Homo sapiens Cell culture supernatant Filtration
Commercial method
Stephanie Jung 2020 0%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
0% (median: 22% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
HDV infected
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
Filtration + Commercial method
Protein markers
EV: TNF alpha
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
HDV infected
EV-producing cells
Huh7-NTC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
ELISA
Detected EV-associated proteins
TNF alpha
NA
EV200032 3/11 Homo sapiens Cell culture supernatant Filtration
Commercial method
Stephanie Jung 2020 0%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
0% (median: 22% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
UV irradiated HDV infected
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
Filtration + Commercial method
Protein markers
EV: TNF alpha
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
UV irradiated HDV infected
EV-producing cells
Huh7-NTC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
ELISA
Detected EV-associated proteins
TNF alpha
NA
EV200032 4/11 Homo sapiens Cell culture supernatant Filtration
Commercial method
Stephanie Jung 2020 0%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
0% (median: 22% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
IFN beta treated
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
Filtration + Commercial method
Protein markers
EV: TNF alpha
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
IFN beta treated
EV-producing cells
Huh7-NTC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
ELISA
Detected EV-associated proteins
TNF alpha
NA
EV200032 9/11 Homo sapiens serum Filtration
Commercial method
Stephanie Jung 2020 0%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
0% (median: 13% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
serum
Sample origin
Control
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
Filtration + Commercial method
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
serum
Sample Condition
Control
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
NA
EV200032 10/11 Homo sapiens serum Filtration
Commercial method
Stephanie Jung 2020 0%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
0% (median: 13% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
serum
Sample origin
HDV/HBV coinfected, HDV cured
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
Filtration + Commercial method
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
serum
Sample Condition
HDV/HBV coinfected, HDV cured
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
NA
EV200032 11/11 Homo sapiens serum Filtration
Commercial method
Stephanie Jung 2020 0%

Study summary

Full title
All authors
Stephanie Jung, Sebastian M Altstetter, Florian Wilsch, Mikhail Shein, Anne K Schütz, Ulrike Protzer
Journal
Matters
Abstract
Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins f (show more...)Hepatitis D Virus (HDV) is a satellite virus requiring a Hepatitis B Virus (HBV) envelope proteins for productive infection. Hepatitis D is the most severe form of viral hepatitis and is a global health threat affecting 15 to 20 million humans. In contrast to the Hepatitis B Virus mono-infection, against which only a minor innate immune response is mounted at most, HBV-HDV coinfection is characterized by strong activation of innate immune responses. To shed light on poorly understood mechanisms of HDV-triggered disease progression, we focussed on the question how immune cells may be activated by HDV. We hypothesized that extracellular vesicles (EVs) released from infected cells mediate this activation. We, therefore, purified EVs from the supernatant of HDV-infected and non-infected cells and incubated them with human peripheral blood mononuclear cells (PBMC) and macrophages. Here we show for the first time that HDV infection leads to the production of EVs which subsequently mediate a proinflammatory cytokine response in primary human immune cells. These data might help to understand how HDV can be sensed by non-infected immune cells. (hide)
EV-METRIC
0% (median: 13% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
serum
Sample origin
HDV/HBV coinfected, HDV positif
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
Filtration + Commercial method
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
serum
Sample Condition
HDV/HBV coinfected, HDV positif
Separation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
qEV
NA
EV190070 1/1 Zingiber officinale Ginger aqueous extract Polyethylene glycol precipitation Kalarikkal SP 2020 0%

Study summary

Full title
All authors
Kalarikkal SP, Prasad D, Kasiappan R, Chaudhari SR, Sundaram GM.
Journal
Sci Rep
Abstract
Edible nanoparticles (ENPs) are nano-sized vesicles derived from edible plants. These ENPs are loade (show more...)Edible nanoparticles (ENPs) are nano-sized vesicles derived from edible plants. These ENPs are loaded with plant derived microRNAs, protein, lipids and phytochemicals. Recently, ginger derived ENPs was shown to prevent inflammatory bowel diseases and colon cancer, in vivo, highlighting their therapeutic potential. Conventionally, differential centrifugation with an ultra-centrifugation step is employed to purify these ENPs which imposes limitation on the cost-effectiveness of their purification. Herein, we developed polyethylene glycol-6000 (PEG6000) based ginger ENP purification (PEG-ENPs) method, which eliminates the need for expensive ultracentrifugation. Using different PEG6000 concentrations, we could recover between 60% to 90% of ENPs compared to ultracentrifugation method. PEG-ENPs exhibit near identical size and zeta potential similar to ultra-ENPs. The biochemical composition of PEG-ENPs, such as proteins, lipids, small RNAs and bioactive content is comparable to that of ultra-ENPs. In addition, similar to ultra-ENPs, PEG-ENPs are efficiently taken up by the murine macrophages and protects cells from hydrogen peroxide induced oxidative stress. Since PEG has been approved as food additive, the PEG method described here will provide a cost-effective alternative to purify ENPs, which can be directly used as a dietary supplement in therapeutic formulations. (hide)
EV-METRIC
0% (median: 0% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Ginger aqueous extract
Sample origin
Control condition
Focus vesicles
Other / edible nanoparticles
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
Polyethylene glycol precipitation
Protein markers
EV:
non-EV:
Proteomics
no
Show all info
Study aim
Function/New methodological development
Sample
Species
Zingiber officinale
Sample Type
Ginger aqueous extract
Sample Condition
Control condition
Separation Method
Other
Name other separation method
Polyethylene glycol precipitation
Protein Concentration Method
Bradford
Other 1
Detected EV-associated proteins
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
1
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NA
DLS
Report type
Size range/distribution
Reported size (nm)
400
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