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You searched for: 2023 (Year of publication)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210141 2/5 Homo sapiens human umbilical vein endothelial cells IAF
Ultrafiltratrion
(d)(U)C
DG
Zhao F 2023 89%

Study summary

Full title
All authors
Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G
Journal
EMBO J
Abstract
Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
ZIKV infected cells
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Immunoaffinity capture (non-commercial)
Ultrafiltratrion
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9
non-EV: Capsid/ E/ LC3/ Calnexin
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human umbilical vein endothelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
2,00E+08
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 performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0%
Highest density fraction
80%
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
P55ST
Speed (g)
250000
Duration (min)
1080
Fraction volume (mL)
0,3
Ultra filtration
Cut-off size (kDa)
100 kDa
Membrane type
Regenerated cellulose
Immunoaffinity capture
Selected surface protein(s)
CD9
Other
Name other separation method
Ultrafiltratrion
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81
Detected contaminants
Capsid/ E
Not detected contaminants
LC3/ Calnexin
Characterization: RNA analysis
RNA analysis
Type
RT(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Wide-field
Report size (nm)
100
EV210141 3/5 Homo sapiens human umbilical vein endothelial cells Ultrafiltratrion
(d)(U)C
DG
Zhao F 2023 89%

Study summary

Full title
All authors
Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G
Journal
EMBO J
Abstract
Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
ZIKV infected cells
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltratrion
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9
non-EV: Capsid/ E/ LC3/ Calnexin
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human umbilical vein endothelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
2,00E+08
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 performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0%
Highest density fraction
80%
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
P55ST
Speed (g)
250000
Duration (min)
1080
Fraction volume (mL)
0,3
Ultra filtration
Cut-off size (kDa)
100 kDa
Membrane type
Regenerated cellulose
Other
Name other separation method
Ultrafiltratrion
Characterization: Protein analysis
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81
Detected contaminants
Capsid/ E
Not detected contaminants
LC3/ Calnexin
Characterization: RNA analysis
RNA analysis
Type
RT(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
100
EV210215 3/4 Homo sapiens PBS spiked with recombinant EV (gag-EGFP HEK293T) DG Van Dorpe S 2023 88%

Study summary

Full title
All authors
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A
Journal
J Nanobiotechnology
Abstract
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide)
EV-METRIC
88% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
PBS spiked with recombinant EV (gag-EGFP HEK293T)
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Protein markers
EV: TSG101/ CD81/ Alix/ p24/ CD9/ syntenin-1
non-EV: None
Proteomics
no
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
PBS spiked with recombinant EV (gag-EGFP HEK293T)
Separation Method
Density gradient
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
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Concentration Method
Not determined
Protein Yield (µg)
as percentage of spiked rEV
ELISA
Antibody details provided?
No
Detected EV-associated proteins
p24
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
140
EV concentration
Yes
Particle yield
as percentage of spiked rEV
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Not reported
EV210215 4/4 Homo sapiens PBS spiked with recombinant EV (gag-EGFP HEK293T) DG Van Dorpe S 2023 88%

Study summary

Full title
All authors
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A
Journal
J Nanobiotechnology
Abstract
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide)
EV-METRIC
88% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
PBS spiked with recombinant EV (gag-EGFP HEK293T)
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Protein markers
EV: TSG101/ CD81/ Alix/ p24/ CD9/ syntenin-1
non-EV: None
Proteomics
no
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
PBS spiked with recombinant EV (gag-EGFP HEK293T)
Separation Method
Density gradient
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
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.8
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Not determined
ELISA
Antibody details provided?
No
Detected EV-associated proteins
p24
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
140
EV concentration
Yes
Particle yield
as percentage of spiked rEV
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Not reported
EV210215 1/4 Homo sapiens Blood plasma (d)(U)C
DG
UF
SEC (non-commercial)
Van Dorpe S 2023 86%

Study summary

Full title
All authors
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A
Journal
J Nanobiotechnology
Abstract
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide)
EV-METRIC
86% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Breast 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
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Albumin/ ApoA1/ ApoB
Proteomics
yes
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
No
Density gradient
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
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange Consortium
Detected contaminants
Albumin/ ApoA1/ ApoB
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV210215 2/4 Homo sapiens urine (d)(U)C
DG
UF
Van Dorpe S 2023 86%

Study summary

Full title
All authors
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A
Journal
J Nanobiotechnology
Abstract
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide)
EV-METRIC
86% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Ultrafiltration
Protein markers
EV: None
non-EV: Albumin/ Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
urine
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
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
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange Consortium
Detected contaminants
Albumin/ Tamm-Horsfall protein
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV210302 1/2 Homo sapiens MKN74 (d)(U)C
Filtration
qEV
Poças J 2023 78%

Study summary

Full title
All authors
Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A
Journal
Proc Natl Acad Sci U S A
Abstract
Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
qEV
Protein markers
EV: Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4
non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MKN74
EV-harvesting Medium
Serum free medium
Cell viability (%)
88
Cell count
73000000
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
Pelleting performed
Yes
Pelleting: time(min)
160
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
160
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Filtration steps
0.2 or 0.22 µm
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4
Not detected contaminants
CytochromeC
Proteomics database
PRIDE Proteomics
Detected contaminants
Calreticulin/ GM130/ PMP70/ Prohibitin
Not detected contaminants
Albumin/ Argonaute2/ CytochromeC/ Tubulin1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
102.1
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.90E+08
EM
EM-type
Transmission-EM/ Immuno-EM
EM protein
SDC4
Image type
Close-up, Wide-field
EV210302 2/2 Homo sapiens MKN74 (d)(U)C
Filtration
qEV
Poças J 2023 78%

Study summary

Full title
All authors
Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A
Journal
Proc Natl Acad Sci U S A
Abstract
Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
SDC4 KO
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
qEV
Protein markers
EV: Alix/ CD9/ CD81/ HSP70/ SDCBP
non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MKN74
EV-harvesting Medium
Serum free medium
Cell viability (%)
92
Cell count
74000000
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
Pelleting performed
Yes
Pelleting: time(min)
160
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
160
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Filtration steps
0.2 or 0.22 µm
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD81/ HSP70/ SDCBP
Not detected contaminants
CytochromeC/ Tubulin1
Proteomics database
PRIDE Proteomics
Detected contaminants
Calreticulin/ GM130/ PMP70/ Prohibitin
Not detected contaminants
Albumin/ Argonaute2/ CytochromeC/ Tubulin1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
86
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 6.75E+08
EM
EM-type
Transmission-EM/ Immuno-EM
EM protein
SDC4
Image type
Close-up, Wide-field
EV210141 4/5 Homo sapiens human umbilical vein endothelial cells Ultrafiltratrion
(d)(U)C
DG
Zhao F 2023 75%

Study summary

Full title
All authors
Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G
Journal
EMBO J
Abstract
Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide)
EV-METRIC
75% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
ZIKV infected cells
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltratrion
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9
non-EV: Capsid/ E
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human umbilical vein endothelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
2,00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
0%
Highest density fraction
80%
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
P55ST
Speed (g)
250000
Duration (min)
1080
Fraction volume (mL)
0,3
Ultra filtration
Cut-off size (kDa)
100 kDa
Membrane type
Regenerated cellulose
Other
Name other separation method
Ultrafiltratrion
Characterization: Protein analysis
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ TSG101/ HSP70/ Syntenin
Detected contaminants
Capsid/ E
Characterization: RNA analysis
RNA analysis
Type
RT(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
EV220412 1/1 Bos taurus ovary-derived granulosa cells (d)(U)C
Filtration
Menjivar, Nico G 2023 67%

Study summary

Full title
All authors
Nico G. Menjivar, Ahmed Gad, Samuel Gebremedhn, Soham Ghosh and Dawit Tesfaye
Journal
Front Cell Dev Biol
Abstract
Climate change-induced global warming results in rises in body temperatures above normal physiologic (show more...)Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes’ thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: CD63/ CD81/ TSG101
non-EV: CYCS
Proteomics
no
Show all info
Study aim
Function/Mechanism of uptake/transfer
Sample
Species
Bos taurus
Sample Type
Cell culture supernatant
EV-producing cells
ovary-derived granulosa cells
EV-harvesting Medium
EV-depleted medium
Cell count
250000
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
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
3.5
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
120000
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81/ TSG101
Not detected contaminants
CYCS
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
137.75
EV concentration
Yes
Particle yield
as numer of particles per mililiter: 1.02E+11
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
137.75
EV220412 2/1 Bos taurus ovary-derived granulosa cells (d)(U)C
Filtration
Menjivar, Nico G 2023 67%

Study summary

Full title
All authors
Nico G. Menjivar, Ahmed Gad, Samuel Gebremedhn, Soham Ghosh and Dawit Tesfaye
Journal
Front Cell Dev Biol
Abstract
Climate change-induced global warming results in rises in body temperatures above normal physiologic (show more...)Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes’ thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Heat stress
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: CD63/ CD81/ TSG101
non-EV: CYCS
Proteomics
no
Show all info
Study aim
Function/Mechanism of uptake/transfer
Sample
Species
Bos taurus
Sample Type
Cell culture supernatant
EV-producing cells
ovary-derived granulosa cells
EV-harvesting Medium
EV-depleted medium
Cell count
250000
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
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
3.5
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
120000
Filtration steps
0.2 or 0.22 µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81/ TSG101
Not detected contaminants
CYCS
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
147.95
EV concentration
Yes
Particle yield
as numer of particles per mililiter: 1.51E+11
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
147.95
EV220409 1/2 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers ME 2023 67%

Study summary

Full title
All authors
Kuipers ME, Nguyen DL, van Diepen A, Mes L, Bos E, Koning RI, Nolte-'t Hoen ENM, Smits HH, Hokke CH
Journal
Front Mol Biosci
Abstract
Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released para (show more...)Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host. (hide)
EV-METRIC
67% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
adult worm
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP2
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.18
Show all info
Study aim
Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
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
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
41.60%
Total gradient volume, incl. sample (mL)
2.113
Sample volume (mL)
0.293
Orientation
Bottom-up
Speed (g)
166,18
Duration (min)
120
Fraction volume (mL)
0.1
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.75
Pelleting: speed (g)
187,813
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
yes, per 100 adult worms
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
S. mansoni TSP2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-250
EV concentration
Yes
Particle yield
number per 100 adult worms
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
30-180
Extra information
All data on protein concentration and NTA of the schistosomula EVs can be found in EV track ID EV190032. More information on the protocol for the cryo EM of this publication can be found in EV track ID EV220119. Furthermore, we performed glycomics, western blots targeting glycan structures, and lectin blots of the adult worm EVs. The western blots targeting glycan structures were also performed on the schistosomula EV (glycomics on these EV are linked to EV track ID EV190032).
EV220366 1/11 Mus musculus bone marrow-derived cells (d)(U)C Sako Y 2023 67%

Study summary

Full title
All authors
Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T
Journal
ACS Chem Biol
Abstract
Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD81/ CD86/ CD80/ MHCII/ CD40
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
bone marrow-derived cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell count
3.00E+07
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 performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
30
Wash: time (min)
180
Wash: Rotor Type
SW 28
Wash: speed (g)
100,000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD81/ CD86/ CD80/ MHCII
Not detected EV-associated proteins
CD40
Detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
75-300
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 5.50E+11
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV220366 3/11 Mus musculus bone marrow-derived cells (d)(U)C Sako Y 2023 67%

Study summary

Full title
All authors
Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T
Journal
ACS Chem Biol
Abstract
Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Ionomycin
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD81/ CD86/ CD80/ MHCII/ CD40
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
bone marrow-derived cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell count
3.00E+07
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 performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
30
Wash: time (min)
180
Wash: Rotor Type
SW 28
Wash: speed (g)
100,000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD81/ CD86/ CD80/ MHCII
Not detected EV-associated proteins
CD40
Detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
75-300
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 7.50E+11
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV220366 4/11 Mus musculus bone marrow-derived cells (d)(U)C Sako Y 2023 67%

Study summary

Full title
All authors
Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T
Journal
ACS Chem Biol
Abstract
Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
compound 634
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD81/ CD86/ CD80/ MHCII/ CD40
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
bone marrow-derived cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell count
3.00E+07
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 performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
30
Wash: time (min)
180
Wash: Rotor Type
SW 28
Wash: speed (g)
100,000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD81/ CD86/ CD80/ MHCII
Not detected EV-associated proteins
CD40
Detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
75-300
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 8.00E+11
EM
EM-type
Transmission­-EM
Image type
Wide-field
EV210141 1/5 Homo sapiens human umbilical vein endothelial cells IAF
Ultrafiltratrion
(d)(U)C
Zhao F 2023 67%

Study summary

Full title
All authors
Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G
Journal
EMBO J
Abstract
Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Immunoaffinity capture (non-commercial)
Ultrafiltratrion
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9
non-EV: Capsid/ E/ LC3/ Calnexin
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human umbilical vein endothelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
2,00E+08
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 performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
100 kDa
Membrane type
Regenerated cellulose
Immunoaffinity capture
Selected surface protein(s)
CD9
Other
Name other separation method
Ultrafiltratrion
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81
Detected contaminants
Capsid/ E
Not detected contaminants
LC3/ Calnexin
Characterization: RNA analysis
RNA analysis
Type
RT(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Wide-field
Report size (nm)
100
EV210077 4/6 Homo sapiens Serum (d)(U)C Lapitz A 2023 67%

Study summary

Full title
All authors
Lapitz A, Azkargorta M, Milkiewicz P, Olaizola P, Zhuravleva E, Grimsrud MM, Schramm C, Arbelaiz A, O'Rourke CJ, La Casta A, Milkiewicz M, Pastor T, Vesterhus M, Jimenez-Agüero R, Dill MT, Lamarca A, Valle JW, Macias RIR, Izquierdo-Sanchez L, Castaño YP, Caballero-Camino FJ, Riaño I, Krawczyk M, Ibarra C, Bustamante J, Nova-Camacho LM, Falcon-Perez JM, Elortza F, Perugorria MJ, Andersen JB, Bujanda L, Karlsen TH, Folseraas T, Rodrigues PM, Banales JM
Journal
J Hepatol
Abstract
Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early d (show more...)Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early diagnostic methods, especially important for individuals at high-risk (i.e., primary sclerosing cholangitis (PSC)). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). (hide)
EV-METRIC
67% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
CCA
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ CRP/ OIT3
non-EV: GRP78
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
3
Wash: time (min)
75
Wash: Rotor Type
TLA-110
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81/ FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ CRP/ OIT3
Not detected contaminants
GRP78
Proteomics database
Yes: PRIDE. Orbitrap cohort: P
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
198
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV230062 5/6 Mus musculus Blood plasma (d)(U)C
Filtration
Choi YY 2023 63%

Study summary

Full title
All authors
Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM
Journal
J Extracell Vesicles
Abstract
People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide)
EV-METRIC
63% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: CD9/ CD63/ CD81/ CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41
non-EV: Calnexin/ GM130
Proteomics
no
Show all info
Study aim
Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Between 0.22 and 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
Not reported
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Detected contaminants
Calnexin/ GM130
Flow cytometry
Type of Flow cytometry
conventional flow cytometry
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
Antibody details provided?
No
Not detected EV-associated proteins
CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ RNA -sequencing
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
85
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
conventional flow cytometry
Hardware adjustment
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: ~35000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220323 2/2 Homo sapiens Serum exoEasy Kim JA 2023 63%

Study summary

Full title
All authors
Kim JA, Park C, Sung JJ, Seo DJ, Choi SJ, Hong YH
Journal
Sci Rep
Abstract
Dysregulation of microRNAs (miRNA) in small extracellular vesicles (sEV) such as exosomes have been (show more...)Dysregulation of microRNAs (miRNA) in small extracellular vesicles (sEV) such as exosomes have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Although circulating cell-free miRNA have been extensively investigated in ALS, sEV-derived miRNAs have not been systemically explored yet. Here, we performed small RNA sequencing analysis of serum sEV and identified 5 differentially expressed miRNA in a discovery cohort of 12 patients and 11 age- and sex-matched healthy controls (fold change > 2, p < 0.05). Two of them (up- and down-regulation of miR-23c and miR192-5p, respectively) were confirmed in a separate validation cohort (18 patients and 15 healthy controls) by droplet digital PCR. Bioinformatic analysis revealed that these two miRNAs interact with distinct sets of target genes and involve biological processes relevant to the pathomechanism of ALS. Our results suggest that circulating sEV from ALS patients have distinct miRNA profiles which may be potentially useful as a biomarker of the disease. (hide)
EV-METRIC
63% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Amyotrophic lateral sclerosis
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
exoEasy
Protein markers
EV: CD63/ CD81
non-EV: Calnexin/ GM130
Proteomics
no
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
Commercial kit
exoEasy
Other
Name other separation method
exoEasy
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD81
Not detected contaminants
Calnexin/ GM130
Characterization: RNA analysis
RNA analysis
Type
RNA-sequencing/ droplet digital PCR
Database
Vesiclepedia
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
138
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
138
EV220407 1/1 Homo sapiens adult primary astrocytes (d)(U)C Shanthi KB 2023 57%

Study summary

Full title
All authors
Shanthi KB, Fischer D, Sharma A, Kiviniemi A, Kaakinen M, Vainio SJ, Bart G
Journal
Genes (Basel)
Abstract
Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and (show more...)Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin β1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs. (hide)
EV-METRIC
57% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD9/ CD63/ CD81/ Syntenin
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
adult primary astrocytes
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 performed
Yes
Pelleting: rotor type
AH-629 (36 ml)
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Other 2
Exoview
Detected EV-associated proteins
CD9/ CD63/ CD81/ Syntenin
Characterization: RNA analysis
RNA analysis
Type
RNA-sequencing
Proteinase treatment
Yes
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
660 µg/ml
RNAse treatment
Yes
RNAse type
RNase A and RNase T1
RNAse concentration
0.5 mg/ml
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
107.9
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.20E+06
EM
EM-type
Transmission-EM/ Immuno-EM
EM protein
Integrin beta1
Image type
Close-up, Wide-field
Report size (nm)
50 to 200 nm
EV concentration
Non
EV230567 1/2 Homo sapiens Blood plasma (d)(U)C Li L 2023 56%

Study summary

Full title
All authors
Li L, Li F, Bai X, Jia H, Wang C, Li P, Zhang Q, Guan S, Peng R, Zhang S, Dong JF, Zhang J, Xu X
Journal
Pharmacol Res
Abstract
Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury ( (show more...)Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury (TBI). We previously demonstrated that extracellular vesicles (EVs) released from injured brains led to endothelial barrier disruption and vascular leakage. However, the molecular mechanisms of this EV-induced endothelial dysfunction (endotheliopathy) remain unclear. Here, we enriched plasma EVs from TBI patients (TEVs), and detected high mobility group box 1 (HMGB1) exposure to 50.33 ± 10.17% of TEVs and the number of HMGB1TEVs correlated with injury severity. We then investigated for the first time the impact of TEVs on endothelial function using adoptive transfer models. We found that TEVs induced dysfunction of cultured human umbilical vein endothelial cells and mediated endothelial dysfunction in both normal and TBI mice, which were propagated through the HMGB1-activated receptor for advanced glycation end products (RAGE)/Cathepsin B signaling, and the resultant NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and canonical caspase-1/gasdermin D (GSDMD)-dependent pyroptosis. Finally, von Willebrand factor (VWF) was detected on the surface of 77.01 ± 7.51% of HMGB1TEVs. The TEV-mediated endotheliopathy was reversed by a polyclonal VWF antibody, indicating that VWF might serve a coupling factor that tethered TEVs to ECs, thus facilitating HMGB1-induced endotheliopathy. These results suggest that circulating EVs isolated from patients with TBI alone are sufficient to induce endothelial dysfunction and contribute to secondary brain injury that are dependent on immunologically active HMGB1 exposed on their surface. This finding provided new insight for the development of potential therapeutic targets and diagnostic biomarkers for TBI. (hide)
EV-METRIC
56% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD9/ CD81/ TSG101/ Calnexin
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
0.1
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ TSG101
Not detected EV-associated proteins
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
119.2±10.09
Particle analysis: flow cytometry
Flow cytometer type
FACS LSR II flow cytometer
Hardware adjustment
-
Calibration bead size
0.5/ 0.9/ 3
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 5.32±1.82E07 and 2.4 ± 0.69E07
EM
EM-type
Transmission-EM
Image type
Close-up
EV230567 2/2 Homo sapiens Blood plasma (d)(U)C Li L 2023 56%

Study summary

Full title
All authors
Li L, Li F, Bai X, Jia H, Wang C, Li P, Zhang Q, Guan S, Peng R, Zhang S, Dong JF, Zhang J, Xu X
Journal
Pharmacol Res
Abstract
Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury ( (show more...)Endothelial dysfunction is a key proponent of pathophysiological process of traumatic brain injury (TBI). We previously demonstrated that extracellular vesicles (EVs) released from injured brains led to endothelial barrier disruption and vascular leakage. However, the molecular mechanisms of this EV-induced endothelial dysfunction (endotheliopathy) remain unclear. Here, we enriched plasma EVs from TBI patients (TEVs), and detected high mobility group box 1 (HMGB1) exposure to 50.33 ± 10.17% of TEVs and the number of HMGB1TEVs correlated with injury severity. We then investigated for the first time the impact of TEVs on endothelial function using adoptive transfer models. We found that TEVs induced dysfunction of cultured human umbilical vein endothelial cells and mediated endothelial dysfunction in both normal and TBI mice, which were propagated through the HMGB1-activated receptor for advanced glycation end products (RAGE)/Cathepsin B signaling, and the resultant NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and canonical caspase-1/gasdermin D (GSDMD)-dependent pyroptosis. Finally, von Willebrand factor (VWF) was detected on the surface of 77.01 ± 7.51% of HMGB1TEVs. The TEV-mediated endotheliopathy was reversed by a polyclonal VWF antibody, indicating that VWF might serve a coupling factor that tethered TEVs to ECs, thus facilitating HMGB1-induced endotheliopathy. These results suggest that circulating EVs isolated from patients with TBI alone are sufficient to induce endothelial dysfunction and contribute to secondary brain injury that are dependent on immunologically active HMGB1 exposed on their surface. This finding provided new insight for the development of potential therapeutic targets and diagnostic biomarkers for TBI. (hide)
EV-METRIC
56% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Traumatic brain injury
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD9/ CD81/ TSG101/ Calnexin
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
0.1
Wash: time (min)
60
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ TSG101
Not detected EV-associated proteins
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
118.4±6.98
Particle analysis: flow cytometry
Flow cytometer type
FACS LSR II flow cytometer
Hardware adjustment
nanoscale flow cytometry
Calibration bead size
0.5/ 0.9/ 3
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.4±0.69E07
EM
EM-type
Transmission-EM
Image type
Close-up
EV230053 1/2 Homo sapiens Blood plasma (d)(U)C Bettio V 2023 56%

Study summary

Full title
All authors
Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulati (show more...)Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery. (hide)
EV-METRIC
56% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD9/ CD63/ CD81/ HSP70/ MHC1
non-EV: Histones/ Albumin/ APOB48/B100/ APOA1
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
146
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ HSP70
Detected contaminants
Albumin/ APOB48/B100/ APOA1
Not detected contaminants
Histones
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ MHC1
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
No
Selected surface protein(s)
CD9/ CD63/ CD81
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-170
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1000000-25000000
EV230053 2/2 Homo sapiens Blood plasma (d)(U)C Bettio V 2023 56%

Study summary

Full title
All authors
Bettio V, Mazzucco E, Antona A, Cracas S, Varalda M, Venetucci J, Bruno S, Chiabotto G, Venegoni C, Vasile A, Chiocchetti A, Quaglia M, Camussi G, Cantaluppi V, Panella M, Rolla R, Manfredi M, Capello D
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulati (show more...)Extracellular vesicles (EVs) isolated from plasma are increasingly recognized as promising circulating biomarkers for disease discovery and progression, as well as for therapeutic drug delivery. The scientific community underlined the necessity of standard operative procedures for the isolation and storage of the EVs to ensure robust results. The understanding of the impact of the pre-analytical variables is still limited and some considerations about plasma anticoagulants and isolation methods are necessary. Therefore, we performed a comparison study between EVs isolated by ultracentrifugation and by affinity substrate separation from plasma EDTA and sodium citrate. The EVs were characterized by Nano Tracking Analysis, Western Blot, cytofluorimetric analysis of surface markers, and lipidomic analysis. While anticoagulants did not significantly alter any of the analyzed parameters, the isolation methods influenced EVs size, purity, surface markers expression and lipidomic profile. Compared to ultracentrifugation, affinity substrate separation yielded bigger particles highly enriched in tetraspanins (CD9, CD63, CD81), fatty acids and glycerolipids, with a predominant LDL- and vLDL-like contamination. Herein, we highlighted that the isolation method should be carefully evaluated prior to study design and the need of standardized operative procedures for EVs isolation and application to biomarkers discovery. (hide)
EV-METRIC
56% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD9/ CD63/ CD81/ HSP70/ MHC1
non-EV: Histones/ Albumin/ APOB48/B100/ APOA1
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
146
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ HSP70
Detected contaminants
Albumin/ APOB48/B100/ APOA1
Not detected contaminants
Histones
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ MHC1
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
No
Selected surface protein(s)
CD9/ CD63/ CD81
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
150-210
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 12000000-68000000
EV220406 1/1 Equus caballus Follicular fluid (d)(U)C
Filtration
Gebremedhn S 2023 56%

Study summary

Full title
All authors
Gebremedhn S, Gad A, Ishak GM, Menjivar NG, Gastal MO, Feugang JM, Prochazka R, Tesfaye D, Gastal EL
Journal
Mol Hum Reprod
Abstract
Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerab (show more...)Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerable utility of the mare to study aspects of follicular dynamics and reproductive function in view of the largely constricted, human research subjects. The bi-directional communication between the growing oocyte and the surrounding somatic cells embodies the hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) encapsulated with microRNAs (miRNAs) and present in the follicular fluid (FF). Here, we aimed to decipher the dynamics of the miRNAs in EVs from equine FF aspirated in vivo during different stages of follicular development, namely, predeviation (PreDev/ 18-20 mm), deviation (Dev/ 22-25 mm), postdeviation (PostDev/ 26-29 mm), preovulatory (PreOV/ 30-35 mm), and impending ovulation (IMP/ ∼40 mm). Approximately 176 known miRNAs were found in all groups with 144 mutually detected among all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in expression from the PreDev until the PreOV stage. Another cluster of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression pattern at the PreDev stage until the PostDev stage, with a significant increase at the PreOV stage followed by a significant decrease at the IMP stage. In conclusion, this study provides greater insights into the stage-specific expression dynamics of FF EV-miRNAs during equine follicular development, which may propose novel approaches to improve ART and provide new biomarkers to facilitate the assessment of ovarian pathophysiological conditions. (hide)
EV-METRIC
56% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Follicular fluid
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: CD63/ TSG101/ Flotillin-1
non-EV: CytC
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Equus caballus
Sample Type
Follicular fluid
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 performed
Yes
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120,000
Wash: volume per pellet (ml)
2
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
120,000
Filtration steps
0.2 or 0.22 ?m
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ TSG101/ Flotillin-1
Not detected contaminants
CytC
Characterization: RNA analysis
RNA analysis
Type
RNA-sequencing
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.50E+08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
91-111
EV230005 1/4 Homo sapiens Serum (d)(U)C Dobra G 2023 55%

Study summary

Full title
All authors
Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K
Journal
Cancers (Basel)
Abstract
Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide)
EV-METRIC
55% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD5L/ MMP-9
non-EV: calnexin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
T-1270
Pelleting: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
Alix/ CD5L
Not detected contaminants
calnexin
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
MMP-9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
77.3
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.44E+12
EM
EM-type
Transmission-EM
Image type
Close-up
EV220319 3/6 Homo sapiens Serum (d)(U)C Schmoldt A 2023 55%

Study summary

Full title
All authors
Małgorzata S. Małys, Maximilian C. Köller, Kristin Papp, Christof Aigner, Daffodil Dioso, Patrick Mucher, Helga Schachner, Michael Bonelli, Helmuth Haslacher, Andrew J. Rees, Renate Kain
Journal
Journal of Extracellular Biology
Abstract
Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers (show more...)Small extracellular vesicles (sEV) purified from blood have great potential clinically as biomarkers for systemic disease; however interpretation is complicated by release of sEV ex vivo after blood taking. To quantify the problem and devise ways to minimise it, we characterised sEV in paired serum, plasma and platelet poor plasma (PPP) samples from healthy donors. Immunoblotting showed twofold greater abundance of CD9 in sEV fractions from fresh serum than from fresh plasma or PPP. MACSPlex confirmed this, and showed that proteins expressed on platelet sEV, either exclusively (CD41b, CD42a and CD62P) or more widely (HLA-ABC, CD24, CD29 and CD31) were also twofold more abundant; by contrast non-platelet proteins (including CD81) were no different. Storage of plasma (but not serum) increased abundance of platelet and selected leukocyte sEV proteins to at least that of serum, and this could be recapitulated by activating cells in fresh plasma by Ca2+, an effect abrogated in PPP. This suggests that a substantial proportion of sEV in serum and stored plasma were generated ex vivo, which is not the case for fresh plasma or PPP. Thus we provide strategies to minimise ex vivo sEV generation and criteria for identifying those that were present in vivo. (hide)
EV-METRIC
55% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD9/ CD63/ CD81
non-EV: Calnexin/ Albumin/ ApoB100
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
P28S(SRP28SA) Swinging bucket rotor
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
32
Wash: time (min)
120
Wash: Rotor Type
P28S(SRP28SA) Swinging bucket rotor
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63
Not detected EV-associated proteins
CD9/ CD63
Detected contaminants
Albumin
Not detected contaminants
Calnexin
ELISA
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Detected contaminants
ApoB100
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
No
Selected surface protein(s)
CD9/ CD63/ CD81/ MACSPlex exosome human kit
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
161.1
EV concentration
Yes
Particle yield
per ml of purification
EM
EM-type
Immuno-EM
EM protein
CD9
Image type
Close-up
EV230062 1/6 Homo sapiens HUVEC/THP1 (coculture) (d)(U)C
Filtration
UF
Choi YY 2023 50%

Study summary

Full title
All authors
Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM
Journal
J Extracell Vesicles
Abstract
People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Protein markers
EV: CD31/ CD105/ CD144/ CD146
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC/THP1 (coculture)
EV-harvesting Medium
Serum-containing medium
Cell viability (%)
90
Cell count
4000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Between 0.22 and 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Flow cytometry
Type of Flow cytometry
conventional flow cytometer
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
Antibody details provided?
No
Not detected EV-associated proteins
CD31/ CD105/ CD144/ CD146
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
119
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
conventional flow cytometry
Hardware adjustment
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: ~2.5E06
EM
EM-type
Transmission-EM
Image type
Wide-field
EV230062 2/6 Homo sapiens HUVEC/THP1 (coculture) (d)(U)C
Filtration
UF
Choi YY 2023 50%

Study summary

Full title
All authors
Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM
Journal
J Extracell Vesicles
Abstract
People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Irradiation of co-culture (1 Gy)
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Ultrafiltration
Protein markers
EV: CD31/ CD105/ CD144/ CD146
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC/THP1 (coculture)
EV-harvesting Medium
Serum-containing medium
Cell viability (%)
90
Cell count
4000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Between 0.22 and 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Flow cytometry
Type of Flow cytometry
conventional flow cytometry
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
Antibody details provided?
No
Not detected EV-associated proteins
CD31/ CD105/ CD144/ CD146
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
112
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
conventional flow cytometry
Hardware adjustment
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: ~5E06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230062 3/6 Homo sapiens Blood plasma (d)(U)C
Filtration
Choi YY 2023 50%

Study summary

Full title
All authors
Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM
Journal
J Extracell Vesicles
Abstract
People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Between 0.22 and 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
Not reported
Flow cytometry
Type of Flow cytometry
conventional flow cytometry
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
Antibody details provided?
No
Not detected EV-associated proteins
CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
82
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
conventional flow cytometry
Hardware adjustment
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: ~32000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230062 4/6 Homo sapiens Blood plasma (d)(U)C
Filtration
Choi YY 2023 50%

Study summary

Full title
All authors
Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM
Journal
J Extracell Vesicles
Abstract
People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Irradiation of blood sample (1 Gy)
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Between 0.22 and 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
Not reported
Flow cytometry
Type of Flow cytometry
conventional flow cytometry
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
Antibody details provided?
No
Not detected EV-associated proteins
CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ HLA-DR/ CD45/ CD41
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
88
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
conventional flow cytometry
Hardware adjustment
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: ~34000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230062 6/6 Mus musculus Blood plasma (d)(U)C
Filtration
Choi YY 2023 50%

Study summary

Full title
All authors
Choi YY, Kim A, Lee Y, Lee YH, Park M, Shin E, Park S, Youn B, Seong KM
Journal
J Extracell Vesicles
Abstract
People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiov (show more...)People exposed to radiation in cancer therapy and nuclear accidents are at increased risk of cardiovascular outcomes in long-term survivors. Extracellular vesicles (EVs) are involved in radiation-induced endothelial dysfunction, but their role in the early stage of vascular inflammation after radiation exposure remains to be fully understood. Herein, we demonstrate that endothelial cell-derived EVs containing miRNAs initiate monocyte activation in radiation-induced vascular inflammation. In vitro co-culture and in vivo experimental data showed that endothelial EVs can be sensitively increased by radiation exposure in a dose-dependent manner, and stimulate monocytes releasing monocytic EVs and adhesion to endothelial cells together with an increase in the expression of genes encoding specific ligands for cell-cell interaction. Small RNA sequencing and transfection using mimics and inhibitors explained that miR-126-5p and miR-212-3p enriched in endothelial EVs initiate vascular inflammation by monocyte activation after radiation exposure. Moreover, miR-126-5p could be detected in the circulating endothelial EVs of radiation-induced atherosclerosis model mice, which was found to be tightly correlated with the atherogenic index of plasma. In summary, our study showed that miR-126-5p and miR-212-3p present in the endothelial EVs mediate the inflammatory signals to activate monocytes in radiation-induced vascular injury. A better understanding of the circulating endothelial EVs content can promote their use as diagnostic and prognostic biomarkers for atherosclerosis after radiation exposure. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Irradiation of whole body (1 Gy)
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41
non-EV: None
Proteomics
no
Show all info
Study aim
Function/Biomarker/Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Between 0.22 and 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
Not reported
Flow cytometry
Type of Flow cytometry
conventional flow cytometry
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
Antibody details provided?
No
Not detected EV-associated proteins
CD31/ CD105/ CD144/ CD146/ CD14/ CD11b/ CD45/ CD41
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ RNA -sequencing
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
71
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
conventional flow cytometry
Hardware adjustment
Calibration bead size
0.22/ 0.45/ 0.88/ 1.35
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: ~39000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220409 2/2 Schistosoma mansoni whole parasite culture (d)(U)C
DG
Kuipers ME 2023 50%

Study summary

Full title
All authors
Kuipers ME, Nguyen DL, van Diepen A, Mes L, Bos E, Koning RI, Nolte-'t Hoen ENM, Smits HH, Hokke CH
Journal
Front Mol Biosci
Abstract
Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released para (show more...)Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host. (hide)
EV-METRIC
50% (21st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
whole parasite culture
Sample origin
schistosomula
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: S. mansoni TSP2
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.18
Show all info
Study aim
Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Schistosoma mansoni
Sample Type
whole parasite culture
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
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
41.60%
Total gradient volume, incl. sample (mL)
2.113
Sample volume (mL)
0.293
Orientation
Bottom-up
Speed (g)
166,18
Duration (min)
120
Fraction volume (mL)
0.1
Fraction processing
Centrifugation
Pelleting: volume per fraction
2.75
Pelleting: speed (g)
187,813
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
S. mansoni TSP2
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
Extra information
All data on protein concentration and NTA of the schistosomula EVs can be found in EV track ID EV190032. More information on the protocol for the cryo EM of this publication can be found in EV track ID EV220119. Furthermore, we performed glycomics, western blots targeting glycan structures, and lectin blots of the adult worm EVs. The western blots targeting glycan structures were also performed on the schistosomula EV (glycomics on these EV are linked to EV track ID EV190032).
EV210077 1/6 Homo sapiens Serum (d)(U)C Lapitz A 2023 45%

Study summary

Full title
All authors
Lapitz A, Azkargorta M, Milkiewicz P, Olaizola P, Zhuravleva E, Grimsrud MM, Schramm C, Arbelaiz A, O'Rourke CJ, La Casta A, Milkiewicz M, Pastor T, Vesterhus M, Jimenez-Agüero R, Dill MT, Lamarca A, Valle JW, Macias RIR, Izquierdo-Sanchez L, Castaño YP, Caballero-Camino FJ, Riaño I, Krawczyk M, Ibarra C, Bustamante J, Nova-Camacho LM, Falcon-Perez JM, Elortza F, Perugorria MJ, Andersen JB, Bujanda L, Karlsen TH, Folseraas T, Rodrigues PM, Banales JM
Journal
J Hepatol
Abstract
Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early d (show more...)Cholangiocarcinoma (CCA), heterogeneous biliary tumors with dismal prognosis, lacks accurate early diagnostic methods, especially important for individuals at high-risk (i.e., primary sclerosing cholangitis (PSC)). Here, we searched for protein biomarkers in serum extracellular vesicles (EVs). (hide)
EV-METRIC
45% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ OIT3
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
3
Wash: time (min)
75
Wash: Rotor Type
TLA-110
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
FGL1/ vWF/ PIGR/ FIBG/ FRIL/ FIBB/ OIT3
Not detected EV-associated proteins
CRP
Proteomics database
Yes: PRIDE. Orbitrap cohort: P
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
215
EV concentration
Yes
EV230005 2/4 Homo sapiens Serum (d)(U)C Dobra G 2023 44%

Study summary

Full title
All authors
Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K
Journal
Cancers (Basel)
Abstract
Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Glioblastoma
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD5L/ MMP-9
non-EV: calnexin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
T-1270
Pelleting: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
Alix/ CD5L
Not detected contaminants
calnexin
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
MMP-9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
80.6
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.46E+12
EV230005 3/4 Homo sapiens Serum (d)(U)C Dobra G 2023 44%

Study summary

Full title
All authors
Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K
Journal
Cancers (Basel)
Abstract
Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Brain metastasis originated from non-small cell lung 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
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD5L/ MMP-9
non-EV: calnexin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
T-1270
Pelleting: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
Alix/ CD5L
Not detected contaminants
calnexin
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
MMP-9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
84.8
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.59E+12
EV230005 4/4 Homo sapiens Serum (d)(U)C Dobra G 2023 44%

Study summary

Full title
All authors
Dobra G, Gyukity-Sebestyén E, Bukva M, Harmati M, Nagy V, Szabó Z, Pankotai T, Klekner Á, Buzás K
Journal
Cancers (Basel)
Abstract
Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell inv (show more...)Matrix metalloproteinase-9 (MMP-9) degrades the extracellular matrix, contributes to tumour cell invasion and metastasis, and its elevated level in brain tumour tissues indicates poor prognosis. High-risk tissue biopsy can be replaced by liquid biopsy/ however, the blood-brain barrier (BBB) prevents tumour-associated components from entering the peripheral blood, making the development of blood-based biomarkers challenging. Therefore, we examined the MMP-9 content of small extracellular vesicles (sEVs)-which can cross the BBB and are stable in body fluids-to characterise tumours with different invasion capacity. From four patient groups (glioblastoma multiforme, brain metastases of lung cancer, meningioma, and lumbar disc herniation as controls), 222 serum-derived sEV samples were evaluated. After isolating and characterising sEVs, their MMP-9 content was measured by ELISA and assessed statistically (correlation, paired -test, Welch's test, ANOVA, ROC). We found that the MMP-9 content of sEVs is independent of gender and age, but is affected by surgical intervention, treatment, and recurrence. We found a relation between low MMP-9 level in sEVs (<28 ppm) and improved survival (8-month advantage) of glioblastoma patients, and MMP-9 levels showed a positive correlation with aggressiveness. These findings suggest that vesicular MMP-9 level might be a useful prognostic marker for brain tumours. (hide)
EV-METRIC
44% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Meningioma
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD5L/ MMP-9
non-EV: calnexin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
T-1270
Pelleting: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
Alix/ CD5L
Not detected contaminants
calnexin
ELISA
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
MMP-9
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
82.7
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 3.61E+12
EV220418 1/8 Mus musculus Heart tissue (d)(U)C Schoger E 2023 44%

Study summary

Full title
All authors
Schoger E, Bleckwedel F, Germena G, Rocha C, Tucholla P, Sobitov I, Möbius W, Sitte M, Lenz C, Samak M, Hinkel R, Varga ZV, Giricz Z, Salinas G, Gross JC, Zelarayán LC
Journal
Commun Biol
Abstract
Aberrant Wnt activation has been reported in failing cardiomyocytes. Here we present single cell tr (show more...)Aberrant Wnt activation has been reported in failing cardiomyocytes. Here we present single cell transcriptome profiling of hearts with inducible cardiomyocyte-specific Wnt activation (β-cat) as well as with compensatory and failing hypertrophic remodeling. We show that functional enrichment analysis points to an involvement of extracellular vesicles (EVs) related processes in hearts of β-cat mice. A proteomic analysis of in vivo cardiac derived EVs from β-cat hearts has identified differentially enriched proteins involving 20 S proteasome constitutes, protein quality control (PQC), chaperones and associated cardiac proteins including α-Crystallin B (CRYAB) and sarcomeric components. The hypertrophic model confirms that cardiomyocytes reacted with an acute early transcriptional upregulation of exosome biogenesis processes and chaperones transcripts including CRYAB, which is ameliorated in advanced remodeling. Finally, human induced pluripotent stem cells (iPSC)-derived cardiomyocytes subjected to pharmacological Wnt activation recapitulated the increased expression of exosomal markers, CRYAB accumulation and increased PQC signaling. These findings reveal that secretion of EVs with a proteostasis signature contributes to early patho-physiological adaptation of cardiomyocytes, which may serve as a read-out of disease progression and can be used for monitoring cellular remodeling in vivo with a possible diagnostic and prognostic role in the future. (hide)
EV-METRIC
44% (35th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method,