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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
EV220024 1/7 Homo sapiens MDA-MB-231 DG
Filtration
UF
SEC (non-commercial)
Roux, Quentin 2023 100%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
EV density (g/ml)
1.09-1.11
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
180000000
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)
16.8
Sample volume (mL)
0.8
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ FGF-2/ Fractalkine/ IL-1RA/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 2/7 Homo sapiens MCF-7 DG
Filtration
UF
SEC (non-commercial)
Roux, Quentin 2023 100%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
EV density (g/ml)
1.09-1.11
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MCF-7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
179999999
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)
16.8
Sample volume (mL)
0.8
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 4/7 Homo sapiens Immortalized patient-derived breast CAF DG
Filtration
UF
SEC (non-commercial)
Roux, Quentin 2023 100%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
EV density (g/ml)
1.09-1.11
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Immortalized patient-derived breast CAF
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Cell count
120000000
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)
16.8
Sample volume (mL)
0.8
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
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
EV220369 1/5 Homo sapiens Blood plasma (d)(U)C Lapin M 2023 78%

Study summary

Full title
All authors
Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O
Journal
PLoS One
Abstract
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control 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/ TSG101
non-EV: ApoA1
Proteomics
no
Show all info
Study aim
Biomarker/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 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
20
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/ TSG101
Detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
1-10000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Equal or larger than 200nm
EV220369 2/5 Homo sapiens Blood plasma (d)(U)C
Filtration
qEV
UF
Lapin M 2023 78%

Study summary

Full title
All authors
Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O
Journal
PLoS One
Abstract
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control 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
Commercial method
Ultrafiltration
Protein markers
EV: CD9/ CD63/ CD81/ TSG101
non-EV: ApoA1
Proteomics
no
Show all info
Study aim
Biomarker/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 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Larger than 0.45 µm
Ultra filtration
Cut-off size (kDa)
100 kDa
Membrane type
Regenerated cellulose
Commercial kit
qEV
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/ TSG101
Detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
1-10000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Below or equal to 200nm
EV220369 3/5 Homo sapiens Blood plasma (d)(U)C
Filtration
ExoEasy
Lapin M 2023 78%

Study summary

Full title
All authors
Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O
Journal
PLoS One
Abstract
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control 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
Commercial method
Protein markers
EV: CD9/ CD63/ CD81/ TSG101
non-EV: ApoA1
Proteomics
no
Show all info
Study aim
Biomarker/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 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Larger than 0.45 µm
Commercial kit
ExoEasy
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/ TSG101
Detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
1-10000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Below or equal to 200nm
EV220369 5/5 Homo sapiens Blood plasma (d)(U)C
Filtration
Lapin M 2023 78%

Study summary

Full title
All authors
Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O
Journal
PLoS One
Abstract
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Pancreatic 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
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker/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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100,000
Filtration steps
Larger than 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ TSG101
Detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
1-10000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Below or equal to 200nm
EV220024 3/7 Homo sapiens Immortalized patient-derived breast CAF (d)(U)C Roux, Quentin 2023 78%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Immortalized patient-derived breast CAF
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Cell count
120000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
60
Wash: Rotor Type
SW 32.1 Ti
Wash: speed (g)
100000
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ TSG101
Detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ PDGF-AA/ IL-8/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 5/7 Homo sapiens Immortalized patient-derived breast CAF Filtration
UF
SEC (non-commercial)
Roux, Quentin 2023 78%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Immortalized patient-derived breast CAF
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Cell count
120000000
Separation Method
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
to complete
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 6/7 Homo sapiens MDA-MB-231 (d)(U)C Roux, Quentin 2023 78%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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/ CD9/ TSG101
non-EV: Argonaute 2
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
180000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
60
Wash: Rotor Type
SW 32.1 Ti
Wash: speed (g)
100000
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Detected contaminants
Argonaute 2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
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% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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
EV230588 1/2 Homo sapiens human umbilical cord mesenchymal stromal cells (d)(U)C
Filtration
SEC (non-commercial)
Tangential flow filtration
Forteza-Genestra MA 2023 75%

Study summary

Full title
All authors
Forteza-Genestra MA, Antich-Rosselló M, Ramis-Munar G, Calvo J, Gayà A, Monjo M, Ramis JM
Journal
Bone Joint Res
Abstract
Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, (show more...)Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. (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
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
Size-exclusion chromatography (non-commercial)
Tangential flow filtration
Protein markers
EV: Alix/ CD9/ CD63/ CD81/ HSC70
non-EV: CytC/ ApoA1
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human umbilical cord mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
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
0.2 or 0.22 ?m
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
5
Resin type
Sephacryl S-400 HR
Other
Name other separation method
Tangential flow filtration
Characterization: Protein analysis
Protein Concentration Method
Absorbance at 280 nm
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ CD81
Not detected EV-associated proteins
HSC70
Not detected contaminants
CytC/ ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230588 2/2 Homo sapiens Platelet lysate (d)(U)C
Filtration
SEC (non-commercial)
Forteza-Genestra MA 2023 75%

Study summary

Full title
All authors
Forteza-Genestra MA, Antich-Rosselló M, Ramis-Munar G, Calvo J, Gayà A, Monjo M, Ramis JM
Journal
Bone Joint Res
Abstract
Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, (show more...)Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. (hide)
EV-METRIC
75% (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
Platelet lysate
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
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD63/ CD81/ HSC70
non-EV: CytC/ ApoA1
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Platelet lysate
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Filtration steps
Larger than 0.45 ?m
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
5
Resin type
Sephacryl S-400 HR
Characterization: Protein analysis
Protein Concentration Method
Absorbance at 280 nm
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63
Not detected EV-associated proteins
CD81/ HSC70
Not detected contaminants
CytC/ ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
121
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230370 7/8 Sus scrofa Primary retinal pigmented epithelial cells DC
DG
(d)(U)C
UF
Hernandez, Belinda J. 2023 75%

Study summary

Full title
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (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
Control condition
Focus vesicles
small 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 cushion
Density gradient
(Differential) (ultra)centrifugation
Ultrafiltration
Protein markers
EV: Syntenin-1/ ANXA2/ ITGB1
non-EV: Calreticulin/ Albumin/ Lamins/ Caspases
Proteomics
yes
EV density (g/ml)
1.07-1.11
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Sus scrofa
Sample Type
Cell culture supernatant
EV-producing cells
Primary retinal pigmented epithelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99
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)
12
Sample volume (mL)
3
Orientation
Bottom-up
Speed (g)
200,000
Duration (min)
240
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: speed (g)
100,000
Density cushion
Density medium
Iodixanol
Sample volume
36
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100,000
Characterization: Protein analysis
Protein Concentration Method
Pierce 660 nm assay
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1/ ANXA2/ ITGB1/ KRT10
Not detected contaminants
Calreticulin
Detected contaminants
Albumin
Not detected contaminants
Lamins/ Caspases
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
125.3
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.20E+06
EV230370 8/8 Sus scrofa Primary retinal pigmented epithelial cells DC
DG
(d)(U)C
UF
Hernandez, Belinda J. 2023 75%

Study summary

Full title
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (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
0.2mM H2O2
Focus vesicles
small 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 cushion
Density gradient
(Differential) (ultra)centrifugation
Ultrafiltration
Protein markers
EV: Syntenin-1/ ANXA2/ ITGB1
non-EV: Calreticulin/ Albumin/ Lamins/ Caspases
Proteomics
yes
EV density (g/ml)
1.07-1.11
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Sus scrofa
Sample Type
Cell culture supernatant
EV-producing cells
Primary retinal pigmented epithelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99
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)
12
Sample volume (mL)
3
Orientation
Bottom-up
Speed (g)
200,000
Duration (min)
240
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: speed (g)
100,000
Density cushion
Density medium
Iodixanol
Sample volume
36
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100,000
Characterization: Protein analysis
Protein Concentration Method
Pierce 660 nm assay
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Syntenin-1/ ANXA2/ ITGB1/ KRT10
Not detected contaminants
Calreticulin
Detected contaminants
Albumin
Not detected contaminants
Lamins/ Caspases
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
143.2
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 3.80E+06
EV230055 3/1 Homo sapiens Brain tissues (d)(U)C
Filtration
qEV
UF
Yiyao Huang 2023 75%

Study summary

Full title
All authors
Yiyao Huang, Tanina Arab, Ashley E. Russell, Emily R. Mallick, Rajini Nagaraj, Evan Gizzie, Javier Redding-Ochoa, Juan C. Troncoso, Olga Pletnikova, Andrey Turchinovich, David A. Routenberg, Kenneth W. Witwer
Journal
Biochem Pharmacol
Abstract
Extracellular vesicles (EVs) are released from different cell types in the central nervous system (C (show more...)Extracellular vesicles (EVs) are released from different cell types in the central nervous system (CNS) and play roles in regulating physiological and pathological functions. Although brain-derived EVs (bdEVs) have been successfully collected from brain tissue, there is not yet a “bdEV Atlas” of EVs from different brain regions. To address this gap, we separated EVs from eight anatomical brain regions of a single individual and subsequently characterized them by count, size, morphology, and protein and RNA content. The greatest particle yield was from cerebellum, while the fewest particles were recovered from the orbitofrontal, postcentral gyrus, and thalamus regions. EV surface phenotyping indicated that CD81 and CD9 were more abundant than CD63 in all regions. Cell-enriched surface markers varied between brain regions. For example, putative neuronal markers NCAM, CD271, and NRCAM were more abundant in medulla, cerebellum, and occipital regions, respectively. These findings, while restricted to tissues from a single individual, suggest that additional studies are warranted to provide more insight into the links between EV heterogeneity and function in the CNS. (hide)
EV-METRIC
75% (83rd 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
Brain tissues
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
Ultrafiltration
Protein markers
EV: Alix/ CD9/ CD63/ CD81/ HCAM/CD44/ CD15/ HLA-DR/DP/DQ/ GD2/ NCAM/ TSPO/ CD36/ CD38/ CD90/Thy1/ CD146/MCAM/ CD29/ CD166/hALCAM/ CD64/ CD307d/ TMEM119/ GD1a/ CD31/PECAM/ CD271/LNGFR/ CD24/ CD40/ CD163/ GJA1/ NRCAM
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Brain tissues
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
TH-641
Pelleting: speed (g)
100000
Filtration steps
0.2 or 0.22 µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63
Not detected contaminants
Calreticulin
Detected EV-associated proteins
CD9/ CD63/ CD81
Detected EV-associated proteins
CD9/ CD63/ CD81/ HCAM/CD44/ CD15/ HLA-DR/DP/DQ/ GD2/ NCAM/ TSPO/ CD36/ CD38/ CD90/Thy1/ CD146/MCAM/ CD29/ CD166/hALCAM/ CD64/ CD307d/ TMEM119/ GD1a/ CD31/PECAM/ CD271/LNGFR/ CD24/ CD40/ CD163/ GJA1/
Characterization: RNA analysis
RNA analysis
Type
RNA-sequencing
Database
GEO
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
Flow Nanoanalyzer (NanoFCM)
Hardware adjustment
Compared with traditional flow cytometry, a smaller flow channel reduces background signal, and lower system pressure increases dwell time of particles for enhanced signal integration.
Calibration bead size
68/ 91/ 113/ 151/ 200
Report type
Size range/distribution
Reported size (nm)
42-137
EV concentration
Yes
Particle yield
2.17-8.95E08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
40-500
EV230013 1/3 Homo sapiens Blood plasma SEC (non-commercial) Darragh IAJ 2023 75%

Study summary

Full title
All authors
Darragh IAJ, McNamee N, Daly R, Pacheco SM, O'Driscoll L, Egan B
Journal
J Physiol
Abstract
Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, (show more...)Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, are released by all cell types and are present in all human biofluids. Changes in EV profiles and abundance occur in response to acute exercise, but this study investigated whether individuals with divergent histories of exercise training (recreationally active controls - CON/ endurance-trained - END/ strength-trained - STR) presented with varied abundances of small EVs in resting samples and whether the abundance of small EVs differed within each group across two measurement days. Participants (n = 38, all male/ CON n = 12, END n = 13, STR n = 13) arrived at the lab on two separate occasions in a rested, overnight fasted state, with standardisation of time of day of sampling, recent dietary intake, time since last meal and time since last exercise training session (∼40 h). Whole blood samples were collected and separated into plasma from which small EVs were separated using size exclusion chromatography and identified in accordance with the Minimal Information For Studies of Extracellular Vesicles (MISEV) guidelines. No differences in the abundance of small EVs were observed within or between groups across multiple methods of small EV identification (nanoparticle tracking analysis, flow cytometry, immunoblot of specific EV markers). Targeted metabolomics of the small EV preparations identified 96 metabolites that were associated with the structure and function of small EVs, with no statistically significant differences in concentrations observed across groups. The results of the current study suggest that the abundance and metabolomic profile of small EVs derived from men with divergent histories of exercise training are similar to those in resting blood samples. KEY POINTS: Extracellular vesicles (EV) are membrane-encapsulated particles that are present in circulation and carry bioactive materials as 'cargo'. The abundance and profile of small EVs are responsive to acute exercise, but little is known about the relationship between small EVs and exercise training. This study examined the abundance, and a targeted metabolomic profile, of small EVs separated from the blood of endurance athletes, strength athletes and recreationally active controls at rest (∼40 h after the most recent exercise session) on two separate but identical lab visits. No differences were observed in the abundance or metabolomic profile of small EV preparations between the groups or between the lab visits within each group. Further research should determine whether the bioactive cargoes (e.g. RNA, protein and additional metabolites) carried within EVs are altered in individuals with divergent histories of exercise training or in response to exercise training interventions. (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
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
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD63/ Flotillin-1/ Alix/ CD9/ TSG101
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Size-exclusion chromatography
Sample volume/column (mL)
1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin-1
Not detected EV-associated proteins
Alix/ CD9/ TSG101
Detected contaminants
Albumin
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD63
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
Yes
Selected surface protein(s)
CD63
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
135
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 75084686.28/mL
Particle analysis: flow cytometry
Flow cytometer type
ImageStream X MK II
Hardware adjustment
60X magnification and low flow rate
Calibration bead size
6
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 4.00E+06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230013 2/3 Homo sapiens Blood plasma SEC (non-commercial) Darragh IAJ 2023 75%

Study summary

Full title
All authors
Darragh IAJ, McNamee N, Daly R, Pacheco SM, O'Driscoll L, Egan B
Journal
J Physiol
Abstract
Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, (show more...)Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, are released by all cell types and are present in all human biofluids. Changes in EV profiles and abundance occur in response to acute exercise, but this study investigated whether individuals with divergent histories of exercise training (recreationally active controls - CON/ endurance-trained - END/ strength-trained - STR) presented with varied abundances of small EVs in resting samples and whether the abundance of small EVs differed within each group across two measurement days. Participants (n = 38, all male/ CON n = 12, END n = 13, STR n = 13) arrived at the lab on two separate occasions in a rested, overnight fasted state, with standardisation of time of day of sampling, recent dietary intake, time since last meal and time since last exercise training session (∼40 h). Whole blood samples were collected and separated into plasma from which small EVs were separated using size exclusion chromatography and identified in accordance with the Minimal Information For Studies of Extracellular Vesicles (MISEV) guidelines. No differences in the abundance of small EVs were observed within or between groups across multiple methods of small EV identification (nanoparticle tracking analysis, flow cytometry, immunoblot of specific EV markers). Targeted metabolomics of the small EV preparations identified 96 metabolites that were associated with the structure and function of small EVs, with no statistically significant differences in concentrations observed across groups. The results of the current study suggest that the abundance and metabolomic profile of small EVs derived from men with divergent histories of exercise training are similar to those in resting blood samples. KEY POINTS: Extracellular vesicles (EV) are membrane-encapsulated particles that are present in circulation and carry bioactive materials as 'cargo'. The abundance and profile of small EVs are responsive to acute exercise, but little is known about the relationship between small EVs and exercise training. This study examined the abundance, and a targeted metabolomic profile, of small EVs separated from the blood of endurance athletes, strength athletes and recreationally active controls at rest (∼40 h after the most recent exercise session) on two separate but identical lab visits. No differences were observed in the abundance or metabolomic profile of small EV preparations between the groups or between the lab visits within each group. Further research should determine whether the bioactive cargoes (e.g. RNA, protein and additional metabolites) carried within EVs are altered in individuals with divergent histories of exercise training or in response to exercise training interventions. (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
Blood plasma
Sample origin
Elite Level Endurance Athletes
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
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD63/ Flotillin-1/ Alix/ CD9/ TSG101
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Size-exclusion chromatography
Sample volume/column (mL)
1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin-1
Not detected EV-associated proteins
Alix/ CD9/ TSG101
Detected contaminants
Albumin
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD63
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
Yes
Selected surface protein(s)
CD63
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
131
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 25937424601/mL
Particle analysis: flow cytometry
Flow cytometer type
ImageStream X MK II
Hardware adjustment
60X magnification and low flow rate
Calibration bead size
6
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.50E+06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230013 3/3 Homo sapiens Blood plasma SEC (non-commercial) Darragh IAJ 2023 75%

Study summary

Full title
All authors
Darragh IAJ, McNamee N, Daly R, Pacheco SM, O'Driscoll L, Egan B
Journal
J Physiol
Abstract
Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, (show more...)Small extracellular vesicles (EV) are membrane-encapsulated particles that carry bioactive cargoes, are released by all cell types and are present in all human biofluids. Changes in EV profiles and abundance occur in response to acute exercise, but this study investigated whether individuals with divergent histories of exercise training (recreationally active controls - CON/ endurance-trained - END/ strength-trained - STR) presented with varied abundances of small EVs in resting samples and whether the abundance of small EVs differed within each group across two measurement days. Participants (n = 38, all male/ CON n = 12, END n = 13, STR n = 13) arrived at the lab on two separate occasions in a rested, overnight fasted state, with standardisation of time of day of sampling, recent dietary intake, time since last meal and time since last exercise training session (∼40 h). Whole blood samples were collected and separated into plasma from which small EVs were separated using size exclusion chromatography and identified in accordance with the Minimal Information For Studies of Extracellular Vesicles (MISEV) guidelines. No differences in the abundance of small EVs were observed within or between groups across multiple methods of small EV identification (nanoparticle tracking analysis, flow cytometry, immunoblot of specific EV markers). Targeted metabolomics of the small EV preparations identified 96 metabolites that were associated with the structure and function of small EVs, with no statistically significant differences in concentrations observed across groups. The results of the current study suggest that the abundance and metabolomic profile of small EVs derived from men with divergent histories of exercise training are similar to those in resting blood samples. KEY POINTS: Extracellular vesicles (EV) are membrane-encapsulated particles that are present in circulation and carry bioactive materials as 'cargo'. The abundance and profile of small EVs are responsive to acute exercise, but little is known about the relationship between small EVs and exercise training. This study examined the abundance, and a targeted metabolomic profile, of small EVs separated from the blood of endurance athletes, strength athletes and recreationally active controls at rest (∼40 h after the most recent exercise session) on two separate but identical lab visits. No differences were observed in the abundance or metabolomic profile of small EV preparations between the groups or between the lab visits within each group. Further research should determine whether the bioactive cargoes (e.g. RNA, protein and additional metabolites) carried within EVs are altered in individuals with divergent histories of exercise training or in response to exercise training interventions. (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
Blood plasma
Sample origin
Elite Level Strength Athletes
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
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD63/ Flotillin-1/ Alix/ CD9/ TSG101
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Size-exclusion chromatography
Sample volume/column (mL)
1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin-1
Not detected EV-associated proteins
Alix/ CD9/ TSG101
Detected contaminants
Albumin
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD63
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
Yes
Selected surface protein(s)
CD63
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
131
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 33945673899/mL
Particle analysis: flow cytometry
Flow cytometer type
ImageStream X MK II
Hardware adjustment
60X magnification and low flow rate
Calibration bead size
6
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.50E+06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220321 8/8 Homo sapiens Blood plasma DG
UF
SEC (non-commercial)
Kashkanova AD 2023 75%

Study summary

Full title
All authors
Kashkanova AD, Blessing M, Reischke M, Baur JO, Baur AS, Sandoghdar V, Van Deun J
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accur (show more...)Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accurate sizing and quantification of EVs remain problematic, given their nanometre size range and small scattering cross-sections. This is compounded by the fact that common EV isolation methods result in co-isolation of particles with comparable features. Especially in blood plasma, similarly-sized lipoproteins outnumber EVs to a great extent. Recently, interferometric nanoparticle tracking analysis (iNTA) was introduced as a particle analysis method that enables determining the size and refractive index of nanoparticles with high sensitivity and precision. In this work, we apply iNTA to differentiate between EVs and lipoproteins, and compare its performance to conventional nanoparticle tracking analysis (NTA). We show that iNTA can accurately quantify EVs in artificial EV-lipoprotein mixtures and in plasma-derived EV samples of varying complexity. Conventional NTA could not report on EV numbers, as it was not able to distinguish EVs from lipoproteins. iNTA has the potential to become a new standard for label-free EV characterization in suspension. (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
Blood plasma
Sample origin
Melanoma
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
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD63/ CD81
non-EV: ApoB
Proteomics
no
EV density (g/ml)
1.1-1.2
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Blood plasma
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)
11.5
Sample volume (mL)
0.5
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-4B
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
ELISA
Antibody details provided?
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Other particle analysis name(1)
interferometric nanoparticle tracking analysis
EV-concentration
Yes
Particle yield
No
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
EV220363 4/4 Rattus norvegicus PC12 (d)(U)C
DG
Tedford E 2023 71%

Study summary

Full title
All authors
Tedford E, Badya NB, Laing C, Asaoka N, Kaneko S, Filippi BM, McConkey GA
Journal
Sci Rep
Abstract
Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammati (show more...)Infection with the protozoan Toxoplasma gondii induces changes in neurotransmission, neuroinflammation, and behavior, yet it remains elusive how these changes come about. In this study we investigated how norepinephrine levels are altered by infection. TINEV (Toxoplasma-induced neuronal extracellular vesicles) isolated from infected noradrenergic cells down-regulated dopamine ß-hydroxylase (DBH) gene expression in human and rodent cells. Here we report that intracerebral injection of TINEVs into the brain is sufficient to induce DBH down-regulation and distrupt catecholaminergic signalling. Further, TINEV treatment induced hypermethylation upstream of the DBH gene. An antisense lncRNA to DBH was found in purified TINEV preparations. Paracrine signalling to induce transcriptional gene silencing and DNA methylation may be a common mode to regulate neurologic function. (hide)
EV-METRIC
71% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Toxoplasma gondii Prugniaud-infected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: CD63/ CD81/ Flotillin-1/ TSG101/ EpCAM/ Alix
non-EV: GM130
Proteomics
no
EV density (g/ml)
1.16
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
EV-producing cells
PC12
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
160000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
11
Lowest density fraction
10%
Highest density fraction
70%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1
Orientation
Bottom-up
Speed (g)
70000
Duration (min)
960
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: speed (g)
160000
Pelleting: adjusted k-factor
1.132
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
per milliliter of starting sample
Other 1
Dot Blot SBI
Detected EV-associated proteins
CD63/ CD81/ Flotillin1/ TSG101/ EpCAM
Not detected EV-associated proteins
Alix
Detected contaminants
none
Not detected contaminants
GM130
Characterization: RNA analysis
RNA analysis
Type
RT-PCR
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
EV230965 2/2 Homo sapiens urine (d)(U)C
ExoQuick
Tao W 2023 67%

Study summary

Full title
All authors
Tao W, Wang BY, Luo L, Li Q, Meng ZA, Xia TL, Deng WM, Yang M, Zhou J, Zhang X, Gao X, Li LY, He YD
Journal
Cell Rep Med
Abstract
To construct a urine extracellular vesicle long non-coding RNA (lncRNA) classifier that can detect h (show more...)To construct a urine extracellular vesicle long non-coding RNA (lncRNA) classifier that can detect high-grade prostate cancer (PCa) of grade group 2 or greater and estimate the risk of progression during active surveillance, we identify high-grade PCa-specific lncRNAs by combined analyses of cohorts from TAHSY, TCGA, and the GEO database. We develop and validate a 3-lncRNA diagnostic model (C, being made of AC015987.1, CTD-2589M5.4, RP11-363E6.3) that can detect high-grade PCa. C shows higher accuracy than prostate cancer antigen 3 (PCA3), multiparametric magnetic resonance imaging (mpMRI), and two risk calculators (Prostate Cancer Prevention Trial [PCPT]-RC 2.0 and European Randomized Study of Screening for Prostate Cancer [ERSPC]-RC) in the training cohort (n = 350), two independent cohorts (n = 232/ n = 251), and TCGA cohort (n = 499). In the prospective active surveillance cohort (n = 182), C at diagnosis remains a powerful independent predictor for overall active surveillance progression. Thus, C is a potential biomarker for high-grade PCa and can also serve as a biomarker for improved selection of candidates for active surveillance. (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
urine
Sample origin
prostate cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
ExoQuick
Protein markers
EV: CD9/ CD63/ CD81/ HSP70
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
urine
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
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
5
Wash: time (min)
30
Wash: speed (g)
1500
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ HSP70
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ RNAsequencing/ dd-PCR
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mode
Reported size (nm)
133
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.68E+09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230962 1/6 Bos bovis bovine milk (d)(U)C Colella, Anna P. 2023 67%

Study summary

Full title
All authors
Anna P. Colella, Anuradha Prakash, John J. Miklavcic
Journal
Food Science and Nutrition
Abstract
Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Indu (show more...)Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Industrial processing treatments may affect the amount or bioactivity of EVs intrinsic to bovine milk. We investigated how the content and concentration of EVs were affected by homogenization and thermal processing of raw bovine milk. Raw milk was processed by homogenization, low-temperature (LT) heat, or pasteurization [high-temperature short-time (HTST) and ultra-high-temperature (UHT)] in a pilot processing facility. EVs were isolated from the raw and processed bovine milk using differential ultracentrifugation and quantified using a nanoparticle tracking analyzer. Bovine milk EVs were assessed for total miRNA and protein concentrations standardized to particle count using a fluorometric assay. There were 1.01 × 1010 (±3.30 × 109) EV particles per ml of bovine milk. All industrial processing treatments caused >60% decrease in EV concentration compared to the raw bovine milk. Homogenization and heat treatments independently and additively reduced the content of EVs in bovine milk. The averages of total miRNA/particle and total protein/particle concentrations were elevated threefold by low-temperature heat-processing treatment relative to HTST and UHT pasteurizations. The average diameter of EVs was reduced by 11%–16% by low temperature compared to raw milk (127 ± 13 nm). Homogenization and pasteurization indiscriminately reduce the EV concentration of bovine milk. Smaller EVs with higher protein content resist degradation when processing bovine milk at sub-pasteurization temperature. This new foundational knowledge may contribute to food product development on the preservation of EVs in processed dairy products, including bovine milk-based infant formulas that some newborns are dependent on for adequate growth and development. (hide)
EV-METRIC
67% (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
bovine milk
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/ CD63/ CD81/ Flotillin-1/ TSG101/ ANXA5/ EpCAM/ ICAM1
non-EV: GM130
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Bos bovis
Sample Type
bovine milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
130000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Detected EV-associated proteins
Alix/ CD63/ CD81/ Flotillin-1/ TSG101/ ANXA5/ EpCAM/ ICAM1
Not detected EV-associated proteins
EpCAM
Not detected contaminants
GM130
Characterization: RNA analysis
RNA analysis
Type
fluorometric for total miRNA
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
127
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.01E+10
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV230603 1/4 Bos taurus Blood plasma (d)(U)C
qEV
Filtration
Turner N 2023 67%

Study summary

Full title
All authors
Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD
Journal
J Proteome Res
Abstract
The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (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
Blood plasma
Sample origin
Low-fertility
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
Commercial method
Filtration
Protein markers
EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Bos taurus
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
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
120,000
Filtration steps
0.2 or 0.22 ?m
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ Flotillin-1/ TSG101
Not detected EV-associated proteins
GAPDH
Detected contaminants
Albumin
Proteomics database
No
Detected contaminants
Albumin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230603 2/4 Bos taurus Blood plasma (d)(U)C
qEV
Filtration
Turner N 2023 67%

Study summary

Full title
All authors
Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD
Journal
J Proteome Res
Abstract
The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (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
Blood plasma
Sample origin
High-fertility
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
Commercial method
Filtration
Protein markers
EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Bos taurus
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
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
120,000
Filtration steps
0.2 or 0.22 ?m
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ Flotillin-1/ TSG101
Not detected EV-associated proteins
GAPDH
Detected contaminants
Albumin
Proteomics database
No
Detected contaminants
Albumin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230603 3/4 Bos taurus Blood plasma (d)(U)C
qEV
Turner N 2023 67%

Study summary

Full title
All authors
Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD
Journal
J Proteome Res
Abstract
The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (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
Blood plasma
Sample origin
Low-fertility
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
Commercial method
Protein markers
EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Bos taurus
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
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70.1 Ti
Pelleting: speed (g)
100,000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ Flotillin-1/ TSG101
Not detected EV-associated proteins
GAPDH
Detected contaminants
Albumin
Proteomics database
No
Detected contaminants
Albumin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
125
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230603 4/4 Bos taurus Blood plasma (d)(U)C
qEV
Turner N 2023 67%

Study summary

Full title
All authors
Turner N, Abeysinghe P, Flay H, Meier S, Sadowski P, Mitchell MD
Journal
J Proteome Res
Abstract
The development of biomarkers of fertility could provide benefits for the genetic improvement of dai (show more...)The development of biomarkers of fertility could provide benefits for the genetic improvement of dairy cows. Circulating small extracellular vesicles (sEVs) show promise as diagnostic or prognostic markers since their cargo reflects the metabolic state of the cell of origin/ thus, they mirror the physiological status of the host. Here, we employed data-independent acquisition mass spectrometry to survey the plasma and plasma sEV proteomes of two different cohorts of Young (Peripubertal/ = 30) and Aged (Primiparous/ = 20) dairy cows () of high- and low-genetic merit of fertility and known pregnancy outcomes (ProteomeXchange data set identifier PXD042891). We established predictive models of fertility status with an area under the curve of 0.97 (sEV/ value = 3.302e-07) and 0.95 (plasma/ value = 6.405e-08). Biomarker candidates unique to high-fertility Young cattle had a sensitivity of 0.77 and specificity of 0.67 (* = 0.0287). Low-fertility biomarker candidates uniquely identified in sEVs from Young and Aged cattle had a sensitivity and specificity of 0.69 and 1.0, respectively (*** = 0.0005). Our bioinformatics pipeline enabled quantification of plasma and circulating sEV proteins associated with fertility phenotype. Further investigations are warranted to validate this research in a larger population, which may lead to improved classification of fertility status in cattle. (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
Blood plasma
Sample origin
High-fertility
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
Commercial method
Protein markers
EV: CD9/ CD81/ Flotillin-1/ TSG101/ GAPDH
non-EV: Albumin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Bos taurus
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
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70.1 Ti
Pelleting: speed (g)
100,000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD81/ Flotillin-1/ TSG101
Not detected EV-associated proteins
GAPDH
Detected contaminants
Albumin
Proteomics database
No
Detected contaminants
Albumin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
112
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230602 3/3 Homo sapiens Blood plasma (d)(U)C Benayas, Beatriz 2023 67%

Study summary

Full title
All authors
Beatriz Benayas, Joaquín Morales, Carolina Egea, Pilar Armisén, María Yáñez-Mó
Journal
J Extracell Biol
Abstract
Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. How (show more...)Interest in the use of extracellular vesicles (EVs) as biomarkers of disease is rapidly growing. However, one main unsolved issue in the EV field is finding a technique able to eliminate non-EV contaminants present in biofluid samples in a one-step isolation protocol. Due to the expansion and value of size exclusion chromatography (SEC) as one of the best EV isolation methods, we have tested several agarose resins with different agarose percentages, bead sizes and crosslinking features to optimize EV isolation. For this optimization of SEC, we first employed conditioned media from a melanoma cell culture, a simpler sample in comparison to biological fluids, but which also contains abundant contaminants such as soluble protein and lipoproteins (LPPs). The distinct agaroses and the combinations of resins with different agarose percentages in the same column were tested. Soluble protein, EVs and LPPs levels from the different eluted fractions were quantitated by immunodetection or absorbance measurements. Samples were also analysed by NTA and TEM to verify the yield and the LPP contamination. Different percentages of agarose resins (2%, 4% and 6%) yielded samples with increasing LPP contamination respectively, which was not improved in the columns that combined them. Crosslinking of the agarose did not affect EV isolation yield nor the LPP contamination. In contrast, reducing the bead size greatly improved EV purity. We thus selected 4% Rapid Run Fine agarose beads as the resin that more efficiently isolated EVs with almost no contamination of other particles. Using blood plasma samples, this resin also demonstrated an improved capacity in the isolation of EVs from LPPs in comparison to the agaroses most commonly used in the field and differential ultracentrifugation. (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
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
non-EV: ApoB/ ApoE
Proteomics
no
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
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: rotor type
AH 627
Pelleting: speed (g)
100000
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/ CD81
Not detected contaminants
ApoB/ ApoE
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
120
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 3.00E+09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV230374 2/13 Homo sapiens HEK293T (d)(U)C Levy-Myers R 2023 67%

Study summary

Full title
All authors
Levy-Myers R, Daudelin D, Na CH, Sockanathan S
Journal
Sci Adv
Abstract
Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the (show more...)Extracellular vesicles (EVs) are heterogeneous in size, composition, and function. We show that the six-transmembrane protein glycerophosphodiester phosphodiesterase 3 (GDE3) regulates actin remodeling, a global EV biogenic pathway, to release an EV subtype with distinct functions. GDE3 is necessary and sufficient for releasing EVs containing annexin A1 and GDE3 from the plasma membrane via Wiskott-Aldrich syndrome protein family member 3 (WAVE3), a major regulator of actin dynamics. GDE3 is expressed in astrocytes but not neurons, yet mice lacking GDE3 [ knockout (KO)] have decreased miniature excitatory postsynaptic current (mEPSC) amplitudes in hippocampal CA1 neurons. EVs from cultured wild-type astrocytes restore mEPSC amplitudes in KOs, while EVs from KO astrocytes or astrocytes inhibited for WAVE3 actin branching activity do not. Thus, GDE3-WAVE3 is a nonredundant astrocytic pathway that remodels actin to release a functionally distinct EV subtype, supporting the concept that independent regulation of global EV release pathways differentially regulates EV signaling within the cellular EV landscape. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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: CD63/ CD81/ ANXA1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HEK293T
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
TLA-100.4
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
An