Search > Results

You searched for: 2020 (Year of publication)

Showing 1 - 50 of 72

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
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
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
EV200036 1/16 Homo sapiens Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Commercial method
Juan Antonio Fafián-Labora 2020 78%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
Young donors
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
Density gradient + (Differential) (ultra)centrifugation + Commercial method
Protein markers
EV: Alix/ TSG101/ GSTM2
non-EV: Calnexin/ Actin-beta
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Young donors
EV-producing cells
human skin primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ GSTM2
Not detected contaminants
Calnexin/ Actin-beta
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 3/16 Homo sapiens Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Commercial method
Juan Antonio Fafián-Labora 2020 78%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
Old donors
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
Density gradient + (Differential) (ultra)centrifugation + Commercial method
Protein markers
EV: Alix/ TSG101/ GSTM2
non-EV: Calnexin/ Actin-beta
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Old donors
EV-producing cells
human skin primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ GSTM2
Not detected contaminants
Calnexin/ Actin-beta
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
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
EV200042 1/2 Mus musculus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Density cushion
Filtration
Laura Bouchareychas 2020 75%

Study summary

Full title
All authors
Laura Bouchareychas, Phat Duong, Sergio Covarrubias, Eric Alsop, Tuan Anh Phu, Allen Chung, Michael Gomes, David Wong, Bessie Meechoovet, Allyson Capili, Ryo Yamamoto, Hiromitsu Nakauchi, Michael T McManus, Susan Carpenter, Kendall Van Keuren-Jensen, Robert L Raffai
Journal
Cell Rep
Abstract
Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remai (show more...)Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery. (hide)
EV-METRIC
75% (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
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
Density gradient + (Differential) (ultra)centrifugation + Density cushion + Filtration
Protein markers
EV: Alix/ Flotillin1/ CD9
non-EV: Calnexin/ GM130
Proteomics
no
EV density (g/ml)
1.09
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Bone marrow-derived macrophages (BMDMs)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Density cushion
Density medium
Iodixanol
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ Alix
Not detected contaminants
Calnexin/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
Other;RNase A/T1 Mix
RNAse concentration
0.4
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
75.66
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 2.70E+09
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
EV200012 1/2 Rattus norvegicus Cell culture supernatant (Differential) (ultra)centrifugation Doreen Matthies 2020 67%

Study summary

Full title
All authors
Doreen Matthies, Nathanael Y J Lee, Ian Gatera, H Amalia Pasolli, Xiaowei Zhao, Hui Liu, Deepika Walpita, Zhe Liu, Zhiheng Yu, Maria S Ioannou
Journal
Sci Rep
Abstract
Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been imp (show more...)Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
(Differential) (ultra)centrifugation
Protein markers
EV: tubulin/ Flotillin1/ syntenin
non-EV: gp96
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Primary cortical neurons
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
180
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
300000
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Characterization: Protein analysis
PMID previous EV protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ syntenin
Not detected EV-associated proteins
tubulin
Not detected contaminants
gp96
PMID previous EV particle analysis
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
99.07+/-69.87
EV concentration
Yes
EV200042 2/2 Mus musculus Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Density cushion
Filtration
Laura Bouchareychas 2020 63%

Study summary

Full title
All authors
Laura Bouchareychas, Phat Duong, Sergio Covarrubias, Eric Alsop, Tuan Anh Phu, Allen Chung, Michael Gomes, David Wong, Bessie Meechoovet, Allyson Capili, Ryo Yamamoto, Hiromitsu Nakauchi, Michael T McManus, Susan Carpenter, Kendall Van Keuren-Jensen, Robert L Raffai
Journal
Cell Rep
Abstract
Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remai (show more...)Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery. (hide)
EV-METRIC
63% (91st 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
genetically modified cell line
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
Density gradient + (Differential) (ultra)centrifugation + Density cushion + Filtration
Protein markers
EV: Alix/ Flotillin1
non-EV: Calnexin/ GM130
Proteomics
no
EV density (g/ml)
1.09
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
genetically modified cell line
EV-producing cells
Immortalized bone marrow-derived macrophages (iBMDMs)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Density cushion
Density medium
Iodixanol
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix
Not detected contaminants
Calnexin/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
Other;RNase A/T1 Mix
RNAse concentration
0.4
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
74.58
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 2.40E+09
EV190050 1/1 Mus musculus Cell culture supernatant DG
Filtration
SEC
Size-exclusion chromatography (non-commercial)
Ger J A Arkesteijn 2020 63%

Study summary

Full title
All authors
Ger J A Arkesteijn, Estefanía Lozano-Andrés, Sten F W M Libregts, Marca H M Wauben
Journal
Cytometry A
Abstract
Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of (show more...) Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of choice to rapidly assess heterogeneity of cell populations in suspension. With the research field of extracellular vesicles (EV) rapidly expanding, there is an increased demand to address heterogeneity of EV populations in biological samples. Although flow cytometry would be the ideal technique to do so, the available instruments are in general not equipped to optimally detect the dim light scatter signals generated by submicron-sized particles like EV. Although sideward scatter light and fluorescence are currently used as a threshold signal to identify EV within samples, the forward scatter light (FSC) parameter is often neglected due to the lack of resolution to distinguish EV-related signals from noise. However, after optimization of FSC detection by adjusting the size of the obscuration bar, we recently showed that certain EV-subsets could only be identified based on FSC. This observation made us to further study the possibilities to enhance FSC-detection of submicron-sized particles. By testing differently sized obscuration bars and differently sized pinholes in the focal plane behind the FSC detection lens, we generated a matrix that allowed us to determine which combination resulted in the lowest optical background in terms of numbers of events regarding FSC detection of submicron-sized particles. We found that a combination of an 8-mm obscuration bar and a 200-μm pinhole reduced optical background in a reproducible manner to such extent that it allowed a robust separation of 100-nm polystyrene beads from background signals within the FSC channel, and even allowed thresholding on FSC without the interference of massive background signals when both beads and EV were measured. These technical adaptations thus significantly improved FSC detection of submicron-sized particles and provide an important lead for the further development and design of flow cytometers that aid in detection of submicron-sized particles. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. (hide)
EV-METRIC
63% (91st 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
breast tumor model
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 + Filtration + SEC + Size-exclusion chromatography (non-commercial)
Protein markers
EV: TSG101/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ Flotillin2/ HSP70/ MHC2/ CD9/ MHC1
non-EV:
Proteomics
no
EV density (g/ml)
1.10-1.12
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
breast tumor model
EV-producing cells
4T1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
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)
1091
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45µm > x > 0.22µm,
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ CD63
Not detected EV-associated proteins
HSP90/ HSP70/ MHC1/ CD81/ Flotillin1/ TSG101/ MHC2/ Flotillin2/ Alix
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
139.8
EV concentration
Yes
EV190032 1/2 Schistosoma mansoni Schisostomula (larval stage) culture supernatant dUC Marije E Kuipers 2020 57%

Study summary

Full title
All authors
Marije E Kuipers, Esther N M Nolte-'t Hoen, Alwin J van der Ham, Arifa Ozir-Fazalalikhan, D Linh Nguyen, Clarize M de Korne, Roman I Koning, John J Tomes, Karl F Hoffmann, Hermelijn H Smits, Cornelis H Hokke
Journal
J Extracell Vesicles
Abstract
Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host imm (show more...)Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galβ1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. (hide)
EV-METRIC
57% (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
Schisostomula (larval stage) 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
Adj. k-factor
213.2 (pelleting) / 213.2 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Mechanism of uptake/transfer, Identification of content (omics approaches)
Sample
Species
Schistosoma mansoni
Sample Type
Schisostomula (larval stage) culture supernatant
Sample Condition
Control condition
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)
80
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
213.2
Wash: time (min)
60
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
120000
Wash: adjusted k-factor
213.2
Characterization: Protein analysis
PMID previous EV protein analysis
26443722
Protein Concentration Method
microBCA
Protein Concentration
6
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
30-650
EV concentration
Yes
Particle yield
23300000000
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
35-190;30-715
EV190032 2/2 Schistosoma mansoni Schisostomula (larval stage) culture supernatant dUC Marije E Kuipers 2020 57%

Study summary

Full title
All authors
Marije E Kuipers, Esther N M Nolte-'t Hoen, Alwin J van der Ham, Arifa Ozir-Fazalalikhan, D Linh Nguyen, Clarize M de Korne, Roman I Koning, John J Tomes, Karl F Hoffmann, Hermelijn H Smits, Cornelis H Hokke
Journal
J Extracell Vesicles
Abstract
Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host imm (show more...)Helminths like Schistosoma mansoni release excretory/secretory (E/S) products that modulate host immunity to enable infection. Extracellular vesicles (EVs) are among these E/S products, yet molecular mechanisms and functionality of S. mansoni EV interaction with host immune cells is unknown. Here we demonstrate that EVs released by S. mansoni schistosomula are internalised by human monocyte-derived dendritic cells (moDCs). Importantly, we show that this uptake was mainly mediated via DC-SIGN (CD209). Blocking DC-SIGN almost completely abrogated EV uptake, while blocking mannose receptor (MR, CD206) or dendritic cell immunoreceptor (DCIR, CLEC4A) had no effect on EV uptake. Mass spectrometric analysis of EV glycans revealed the presence of surface N-glycans with terminal Galβ1-4(Fucα1-3)GlcNAc (LewisX) motifs, and a wide array of fucosylated lipid-linked glycans, including LewisX, a known ligand for DC-SIGN. Stimulation of moDCs with schistosomula EVs led to increased expression of costimulatory molecules CD86 and CD80 and regulatory surface marker PD-L1. Furthermore, schistosomula EVs increased expression of IL-12 and IL-10 by moDCs, which was partly dependent on the interaction with DC-SIGN. These results provide the first evidence that glycosylation of S. mansoni EVs facilitates the interaction with host immune cells and reveals a role for DC-SIGN and EV-associated glycoconjugates in parasite-induced immune modulation. (hide)
EV-METRIC
57% (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
Schisostomula (larval stage) 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
Adj. k-factor
213.2 (pelleting) / 83.21 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Mechanism of uptake/transfer, Identification of content (omics approaches)
Sample
Species
Schistosoma mansoni
Sample Type
Schisostomula (larval stage) culture supernatant
Sample Condition
Control condition
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)
65
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
213.2
Wash: time (min)
65
Wash: Rotor Type
TLS-55
Wash: speed (g)
120000
Wash: adjusted k-factor
83.21
Characterization: Protein analysis
PMID previous EV protein analysis
26443722
Protein Concentration Method
microBCA
Protein Concentration
6
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
30-650
EV concentration
Yes
Particle yield
23300000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
35-190
EV200036 5/16 Homo sapiens Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
Progeria patients
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
Density gradient + (Differential) (ultra)centrifugation + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Progeria patients
EV-producing cells
human skin primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 7/16 Homo sapiens Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
Density gradient + (Differential) (ultra)centrifugation + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 9/16 Homo sapiens Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
iRas
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
Density gradient + (Differential) (ultra)centrifugation + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iRas
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 11/16 Homo sapiens Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
iRas+GSTM2
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
Density gradient + (Differential) (ultra)centrifugation + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iRas+GSTM2
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 13/16 Homo sapiens Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
iC+GSTM2
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
Density gradient + (Differential) (ultra)centrifugation + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iC+GSTM2
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 15/16 Homo sapiens Cell culture supernatant Density gradient
(Differential) (ultra)centrifugation
Commercial method
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
mCherry-CD63
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
Density gradient + (Differential) (ultra)centrifugation + Commercial method
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
mCherry-CD63
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
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)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200012 2/2 Rattus norvegicus Cell culture supernatant Ultrafiltration Doreen Matthies 2020 56%

Study summary

Full title
All authors
Doreen Matthies, Nathanael Y J Lee, Ian Gatera, H Amalia Pasolli, Xiaowei Zhao, Hui Liu, Deepika Walpita, Zhe Liu, Zhiheng Yu, Maria S Ioannou
Journal
Sci Rep
Abstract
Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been imp (show more...)Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology. (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
Ultrafiltration
Protein markers
EV: tubulin/ Flotillin1/ syntenin
non-EV: gp96
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Primary cortical neurons
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Characterization: Protein analysis
PMID previous EV protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ syntenin
Not detected EV-associated proteins
tubulin
Not detected contaminants
gp96
PMID previous EV particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
147.82+/-95.72
EV concentration
Yes
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
EV190100 1/10 Homo sapiens Cell culture supernatant ExoQuick Chaoliang Liao 2020 50%

Study summary

Full title
All authors
Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang
Journal
J Pharm Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
CNE1
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
Calnexin
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
21.04-255.6
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
80-100
EV190100 2/10 Homo sapiens Cell culture supernatant ExoQuick Chaoliang Liao 2020 50%

Study summary

Full title
All authors
Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang
Journal
J Pharm Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
CNE1-LMP1
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
Calnexin
NA
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
80-100
EV190100 6/10 Homo sapiens Cell culture supernatant ExoQuick Chaoliang Liao 2020 50%

Study summary

Full title
All authors
Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang
Journal
J Pharm Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HKI
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
Calnexin
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
24.3-199.2
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
80-100
EV190100 8/10 Homo sapiens Cell culture supernatant ExoQuick Chaoliang Liao 2020 50%

Study summary

Full title
All authors
Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang
Journal
J Pharm Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
C666-1
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
Calnexin
Characterization: Particle analysis
NA
NTA
Report type
Size range/distribution
Reported size (nm)
13.54-225.9
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
80-100
EV190091 1/1 Homo sapiens Cell culture supernatant dUC
SEC
Getnet Midekessa 2020 50%

Study summary

Full title
All authors
Getnet Midekessa, Kasun Godakumara, James Ord, Janeli Viil, Freddy Lättekivi, Keerthie Dissanayake, Sergei Kopanchuk, Ago Rinken, Aneta Andronowska, Sourav Bhattacharjee, Toonika Rinken, Alireza Fazeli
Journal
ACS Omega
Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in i (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl2, and AlCl3) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant (p < 0.05) lowering of the ZP. Moreover, the ZP was significantly (p < 0.05) less negative in the presence of ions with higher valency (Al3+/Ca2+) than in the presence of monovalent ones (Na+/K+). Besides, the ZP of EVs became less negative at acidic pH, and vice versa. The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs. (hide)
EV-METRIC
50% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 + SEC
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
JAR ATCC HTB-144
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Size-exclusion chromatography
Total column volume (mL)
10.5
Sample volume/column (mL)
0.5
Resin type
Sepharose 4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD9/ CD63/ HSP70/ CD81
Characterization: Particle analysis
NA
NTA
Report type
Mean
Reported size (nm)
15 - 500
EV concentration
Yes
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Wide-field
Report size (nm)
120 -200
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