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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in Sample type, Isolation method
Experiment number
  • Experiments differ in Sample type, Vesicle type, Culture condition
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  • Experiments differ in Sample type, Vesicle type, Culture condition
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  • Experiments differ in Sample type, Vesicle type, Culture condition
Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
  • Experiments differ in Sample type, Vesicle type
Experiment number
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Experiment number
  • Experiments differ in Sample type, Isolation method
Experiment number
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Experiment number
  • Experiments differ in Sample Origin
Experiment number
  • Experiments differ in Sample type, Sample condition
Experiment number
  • Experiments differ in Species, Sample type
Experiment number
  • Experiments differ in Separation protocol, Sample type
Experiment number
  • Experiments differ in Species, Sample type
Experiment number
  • Experiments differ in Species, Sample type
Experiment number
  • Experiments differ in Species, Sample type
Experiment number
  • Experiments differ in Sample origin
Experiment number
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Experiment number
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Experiment number
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Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV180050 6/6 Homo sapiens Cell culture supernatant dUC
Filtration
Alice Gualerzi 2019 66%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ beta-actin/ Flotillin-1/ CD9
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
liver stem cells
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
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: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Filtration steps
> 0.45 µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, CD81, CD9, Flotillin-1, TSG101, beta-actin
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
PMID previous EV particle analysis
Other
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
184 ± 33
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV180029 1/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
144.8
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 2/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
VCaP
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
88
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV180029 3/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
785.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
178.3
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 4/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
VCaP
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
111
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 5/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
VCaP
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
785.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
122.7
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV180029 6/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
118.7
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 7/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
785.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
148.8
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180029 8/8 Homo sapiens Cell culture supernatant dUC Palviainen, Mari 2019 66%

Study summary

Full title
All authors
Mari Palviainen ORCID Icon, Heikki Saari ORCID Icon, Olli Kärkkäinen ORCID Icon, Jenna Pekkinen, Seppo Auriola, Marjo Yliperttula, Maija Puhka, Kati Hanhineva & Pia R.-M. Siljander
Journal
J Extracell Vesicles
Abstract
One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficie (show more...)One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography–mass spectrometry (LC–MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
785.9 (pelleting) / 89.2 (washing)
Protein markers
EV: CD81/ TSG101/ CD29/ CD9
non-EV: calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
VCaP
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
20000
Pelleting: adjusted k-factor
785.9
Wash: time (min)
120
Wash: Rotor Type
TLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, TSG101, CD29
Not detected contaminants
calnexin
Characterization: Particle analysis
PMID previous EV particle analysis
Electron microscopy
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
121.4
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180021 1/4 Homo sapiens Cell culture supernatant dUC Bachurski, Daniel 2019 66%

Study summary

Full title
All authors
Daniel Bachurski ORCID Icon, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi ORCID Icon, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek & Elke Pogge von Strandmann
Journal
J Extracell Vesicles
Abstract
The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods t (show more...)The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5–134.3) compared to ZetaView (% DTEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
892 (washing)
Protein markers
EV: TSG101/ HSP70/ CD63/ CD9/ CD81
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
L540
EV-harvesting Medium
Serum free medium
Cell viability
95
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
30
Pelleting: speed (g)
10000
Wash: time (min)
30
Wash: Rotor Type
TLA-55
Wash: speed (g)
10000
Wash: adjusted k-factor
892.0
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101
Not detected contaminants
Calnexin
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-500
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-200
Other particle analysis name(1)
ExoView
Report type
Size range/distribution
Report size
50-200
EV-concentration
No
Extra information
EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView
EV180021 2/4 Homo sapiens Serum dUC Bachurski, Daniel 2019 66%

Study summary

Full title
All authors
Daniel Bachurski ORCID Icon, Maximiliane Schuldner, Phuong-Hien Nguyen, Alexandra Malz, Katrin S Reiners, Patricia C Grenzi ORCID Icon, Felix Babatz, Astrid C Schauss, Hinrich P Hansen, Michael Hallek & Elke Pogge von Strandmann
Journal
J Extracell Vesicles
Abstract
The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods t (show more...)The expanding field of extracellular vesicle (EV) research needs reproducible and accurate methods to characterize single EVs. Nanoparticle Tracking Analysis (NTA) is commonly used to determine EV concentration and diameter. As the EV field is lacking methods to easily confirm and validate NTA data, questioning the reliability of measurements remains highly important. In this regard, a comparison addressing measurement quality between different NTA devices such as Malvern’s NanoSight NS300 or Particle Metrix’ ZetaView has not yet been conducted. To evaluate the accuracy and repeatability of size and concentration determinations of both devices, we employed comparative methods including transmission electron microscopy (TEM) and single particle interferometric reflectance imaging sensing (SP-IRIS) by ExoView. Multiple test measurements with nanospheres, liposomes and ultracentrifuged EVs from human serum and cell culture supernatant were performed. Additionally, serial dilutions and freeze-thaw cycle-dependent EV decrease were measured to determine the robustness of each system. Strikingly, NanoSight NS300 exhibited a 2.0–2.1-fold overestimation of polystyrene and silica nanosphere concentration. By measuring serial dilutions of EV samples, we demonstrated higher accuracy in concentration determination by ZetaView (% BIAS range: 2.7–8.5) in comparison with NanoSight NS300 (% BIAS range: 32.9–36.8). The concentration measurements by ZetaView were also more precise (% CV range: 0.0–4.7) than measurements by NanoSight NS300 (% CV range: 5.4–10.7). On the contrary, quantitative TEM imaging indicated more accurate EV sizing by NanoSight NS300 (% DTEM range: 79.5–134.3) compared to ZetaView (% DTEM range: 111.8–205.7), while being equally repeatable (NanoSight NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below 60 nm compared to TEM and SP-IRIS. Taken together, NTA devices differ strongly in their hardware and software affecting measuring results. ZetaView provided a more accurate and repeatable depiction of EV concentration, whereas NanoSight NS300 supplied size measurements of higher resolution. (hide)
EV-METRIC
66% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
892 (washing)
Protein markers
EV: HSP70/ CD63/ CD9
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
30
Pelleting: speed (g)
10000
Wash: time (min)
30
Wash: Rotor Type
TLA-55
Wash: speed (g)
10000
Wash: adjusted k-factor
892.0
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70
Not detected contaminants
Calnexin
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-500
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-500
Other particle analysis name(1)
ExoView
Report type
Size range/distribution
Report size
50-200
EV-concentration
No
Extra information
EV-Track data set is associated with a technical paper comparing different NTA devices assessed by TEM and ExoView
EV180071 1/3 Homo sapiens Blood plasma SEC
UF
Brahmer A 2019 63%

Study summary

Full title
All authors
Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM.
Journal
J Extracell Vesicles
Abstract
Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide)
EV-METRIC
63% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
SEC + UF
Protein markers
EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142
non-EV: / ApoA1
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Ultra filtration
Cut-off size (kDa)
30
Membrane type
Regenerated cellulose
Commercial kit
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD81/ CD9/ TSG101/ Syntenin/ CD41b/ CD63
Not detected EV-associated proteins
Sarcoglycan-alpha
Detected contaminants
ApoA1
Not detected contaminants
Detected EV-associated proteins
CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
106
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180050 3/6 Homo sapiens Cell culture supernatant SEC
dUC
Filtration
Ultrafiltration
UF
Alice Gualerzi 2019 62%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
62% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
SEC + dUC + Filtration + Ultrafiltration + UF
Protein markers
EV: CD81/ Flotillin-1/ CD63
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
bone marrow-derived mesenchymal stem cells
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
> 0.45 µm,
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Not specified
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
2
Resin type
HiPrep 16/60 Sephacryl S-400 HR
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, Flotillin-1
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
PMID previous EV particle analysis
Other
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
247 ± 68
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV180050 4/6 Homo sapiens Cell culture supernatant SEC
dUC
Filtration
Ultrafiltration
UF
Alice Gualerzi 2019 62%

Study summary

Full title
All authors
Alice Gualerzi, Sander Alexander Antonius Kooijmans, Stefania Niada, Silvia Picciolini, Anna Teresa Brini, Giovanni Camussi & Marzia Bedoni
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerativ (show more...)Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application. (hide)
EV-METRIC
62% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
SEC + dUC + Filtration + Ultrafiltration + UF
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ beta-actin/ Flotillin-1/ CD9
non-EV: Calnexin/ Calreticulin
Proteomics
no
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods, Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
liver stem cells
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
> 0.45 µm,
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Not specified
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
2
Resin type
HiPrep 16/60 Sephacryl S-400 HR
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, CD81, CD9, Flotillin-1, TSG101, beta-actin
Not detected contaminants
Calnexin, Calreticulin
Characterization: Particle analysis
PMID previous EV particle analysis
Other
Extra particle analysis
NTA
Report type
Mean
Reported size (nm)
228 ± 50
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
Other particle analysis name(1)
Raman spectroscopy
EV190020 1/3 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Kyuno, Daisuke 2019 57%

Study summary

Full title
All authors
Kyuno D, Zhao K, Schnölzer M, Provaznik J, Hackert T, Zöller M.
Journal
Int J Cancer
Abstract
Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdow (show more...)Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor-exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEX were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX. This article is protected by copyright. All rights reserved. (hide)
EV-METRIC
57% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Protein markers
EV:
non-EV:
Proteomics
yes
EV density (g/ml)
1.15-1.56
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
SW948
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1.28
Fraction processing
Centrifugation
Pelleting: volume per fraction
50
Pelleting: duration (min)
150
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
50
Pelleting-wash: duration (min)
150
Pelleting-wash: speed (g)
Type 45 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
No
EV190020 2/3 Rattus norvegicus Cell culture supernatant DG
dUC
Filtration
Kyuno, Daisuke 2019 57%

Study summary

Full title
All authors
Kyuno D, Zhao K, Schnölzer M, Provaznik J, Hackert T, Zöller M.
Journal
Int J Cancer
Abstract
Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdow (show more...)Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor-exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEX were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX. This article is protected by copyright. All rights reserved. (hide)
EV-METRIC
57% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Protein markers
EV:
non-EV:
Proteomics
yes
EV density (g/ml)
1.15-1.56
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
ASML
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1.28
Fraction processing
Centrifugation
Pelleting: volume per fraction
50
Pelleting: duration (min)
150
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
50
Pelleting-wash: duration (min)
150
Pelleting-wash: speed (g)
Type 45 Ti
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
No
EV180072 1/4 Homo sapiens Cell culture supernatant UF
Total Exosome Isolation
dUC
Fricke F 2019 57%

Study summary

Full title
All authors
Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J.
Journal
Int J Oncol
Abstract
In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide)
EV-METRIC
57% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
UF + Total Exosome Isolation + dUC
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HCT116-TGFBR2
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting: time(min)
60
Pelleting: rotor type
ClickSeal Biocontainment Rotor with Lid; 24 x 1.5/2.0 mL Tubes
Pelleting: speed (g)
21100
Ultra filtration
Cut-off size (kDa)
10000
Membrane type
Polyethersulfone (PES)
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
128
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV180072 2/4 Homo sapiens Cell culture supernatant UF
Total Exosome Isolation
dUC
Fricke F 2019 57%

Study summary

Full title
All authors
Fricke F, Mussack V, Buschmann D, Hausser I, Pfaffl MW, Kopitz J, Gebert J.
Journal
Int J Oncol
Abstract
In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inac (show more...)In colorectal cancer (CRC) with microsatellite instability (MSI), >90% of cases are affected by inactivating frameshift mutations of transforming growth factor β receptor type 2 (TGFBR2). TGFBR2 deficiency is considered to drive MSI tumor progression by abrogating downstream TGF‑β signaling. This pathway can alter the expression of coding and non‑coding RNAs, including microRNAs (miRNAs), which are also present in extracellular vesicles (EVs) as post‑transcriptional modulators of gene expression. In our previous study, it was shown that TGFBR2 deficiency alters the protein composition and function of EVs in MSI tumors. To investigate whether mutant TGFBR2 may also affect the miRNA cargo of EVs, the present study characterized miRNAs in EVs and their parental MSI tumor cells that differed only in TGFBR2 expression status. The HCT116‑TGFBR2 MSI cell line model enables the doxycycline (dox)‑inducible reconstituted expression of TGFBR2 in an isogenic background (‑dox, TGFBR2 deficient; +dox, TGFBR2 proficient). Small RNA sequencing of cellular and EV miRNAs showed that the majority of the miRNAs (263/471; 56%) were shared between MSI tumor cells and their EVs. Exploratory data analysis revealed the TGBFR2‑dependent cluster separation of miRNA profiles in EVs and MSI tumor cells. This segregation appeared to result from two subsets of miRNAs, the expression of which were regulated in a TGFBR2‑dependent manner (EVs: n=10; MSI cells: n=15). In the EV subset, 7/10 miRNAs were downregulated and 3/10 were upregulated by TGFBR2 deficiency. In the cellular subset, 13/15 miRNAs were downregulated and 2/15 miRNAs were upregulated in the TGFBR2‑deficient cells. The present study emphasizes the general overlap of miRNA profiles in MSI tumor cells and their EVs, but also highlights the impact of a single tumor driver mutation on the expression of individual miRNAs, as exemplified by the downregulation of miR‑381‑3p in TGFBR2‑deficient MSI tumor cells and their secreted EVs. (hide)
EV-METRIC
57% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
genetically modified cell line
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
UF + Total Exosome Isolation + dUC
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
genetically modified cell line
EV-producing cells
HCT116-TGFBR2
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting: time(min)
60
Pelleting: rotor type
ClickSeal Biocontainment Rotor with Lid Thermo Scientific
Pelleting: speed (g)
21100
Ultra filtration
Cut-off size (kDa)
10000
Membrane type
Polyethersulfone (PES)
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
Bradford
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
125
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV180060 1/2 Homo sapiens NA UF
Filtration
SEC
FC
dUC
Benedikter BJ 2019 57%

Study summary

Full title
All authors
Benedikter BJ, Bouwman FG, Heinzmann ACA, Vajen T, Mariman EC, Wouters EFM, Savelkoul PHM, Koenen RR, Rohde GGU, van Oerle R, Spronk HM, Stassen FRM
Journal
J Extracell Vesicles
Abstract
Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed (show more...)Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. (hide)
EV-METRIC
57% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
Control condition
Focus vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
UF + Filtration + SEC + FC + dUC
Protein markers
EV: CD63/ MFGE8/ CD81/ TF/ HSP70/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
NA
Sample Condition
Control condition
EV-producing cells
BEAS-2B
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
HSP70/ CD81/ MFGE8/ CD63
Flow cytometry specific beads
Detected EV-associated proteins
TF/ CD63/ CD81/ CD9
Proteomics database
Yes
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
80-250
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
70
EV180060 2/2 Homo sapiens NA UF
Filtration
SEC
FC
dUC
Benedikter BJ 2019 57%

Study summary

Full title
All authors
Benedikter BJ, Bouwman FG, Heinzmann ACA, Vajen T, Mariman EC, Wouters EFM, Savelkoul PHM, Koenen RR, Rohde GGU, van Oerle R, Spronk HM, Stassen FRM
Journal
J Extracell Vesicles
Abstract
Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed (show more...)Airway epithelial cells secrete extracellular vesicles (EVs) under basal conditions and when exposed to cigarette smoke extract (CSE). Getting insights into the composition of these EVs will help unravel their functions in homeostasis and smoking-induced pathology. Here, we characterized the proteomic composition of basal and CSE-induced airway epithelial EVs. BEAS-2B cells were left unexposed or exposed to 1% CSE for 24 h, followed by EV isolation using ultrafiltration and size exclusion chromatography. Isolated EVs were labelled with tandem mass tags and their proteomic composition was determined using nano-LC-MS/MS. Tissue factor (TF) activity was determined by a factor Xa generation assay, phosphatidylserine (PS) content by prothrombinase assay and thrombin generation using calibrated automated thrombogram (CAT). Nano-LC-MS/MS identified 585 EV-associated proteins with high confidence. Of these, 201 were differentially expressed in the CSE-EVs according to the moderated t-test, followed by false discovery rate (FDR) adjustment with the FDR threshold set to 0.1. Functional enrichment analysis revealed that 24 proteins of the pathway haemostasis were significantly up-regulated in CSE-EVs, including TF. Increased TF expression on CSE-EVs was confirmed by bead-based flow cytometry and was associated with increased TF activity. CSE-EVs caused faster and more thrombin generation in normal human plasma than control-EVs, which was partly TF-, but also PS-dependent. In conclusion, proteomic analysis allowed us to predict procoagulant properties of CSE-EVs which were confirmed in vitro. Cigarette smoke-induced EVs may contribute to the increased cardiovascular and respiratory risk observed in smokers. (hide)
EV-METRIC
57% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
1% cigarette smoke extract
Focus vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
UF + Filtration + SEC + FC + dUC
Protein markers
EV: TF/ CD81/ CD63/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
NA
Sample Condition
1% cigarette smoke extract
EV-producing cells
BEAS-2B
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Flow cytometry specific beads
Detected EV-associated proteins
TF/ CD63/ CD81/ CD9
Proteomics database
Yes
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
80-250
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
65
EV180009 3/3 Danio rerio Dissociated embryo IAF
dUC
Frederik J.Verweij 2019 57%

Study summary

Full title
All authors
Frederik J.Verweij, Celine Revenu, Guillaume Arras, Florent Dingli, Damarys Loew, Michiel D.Pegtel, Gautier Follain, Guillaume Allio, Jacky G.Goetz, Pascale Zimmermann, Philippe Herbomel, Filippo Del Bene, GraçaRaposo, Guillaumevan Niel
Journal
Cell Press
Abstract
Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physio (show more...)Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physiological relevance remains challenging due to a lack of appropriate model organisms. Here, we developed an in vivo model to study EV function by expressing CD63-pHluorin in zebrafish embryos. A combination of imaging methods and proteomic analysis allowed us to study biogenesis, composition, transfer, uptake, and fate of individual endogenous EVs. We identified a subpopulation of EVs with exosome features, released in a syntenin-dependent manner from the yolk syncytial layer into the blood circulation. These exosomes are captured, endocytosed, and degraded by patrolling macrophages and endothelial cells in the caudal vein plexus (CVP) in a scavenger receptor- and dynamin-dependent manner. Interference with exosome biogenesis affected CVP growth, suggesting a role in trophic support. Altogether, our work represents a system for studying endogenous EV function in vivo with high spatiotemporal accuracy, demonstrating functional inter-organ communication by exosomes. (hide)
EV-METRIC
57% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Dissociated embryo
Sample origin
Overexpression of CD63-phluorin in yolk syncitial layer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
IAF + dUC
Adj. k-factor
41.45 (pelleting) / 41.45 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function, Biogenesis/cargo sorting, Mechanism of uptake/transfer, New methodological development, Identification of content (omics approaches), Interorgan transfer of EVs in vivo
Sample
Species
Danio rerio
Sample Type
Dissociated embryo
Sample Condition
Overexpression of CD63-phluorin in yolk syncitial layer
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
TLA-120.1
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
41.45
Wash: time (min)
60
Wash: Rotor Type
TLA-120.2
Wash: speed (g)
100000
Wash: adjusted k-factor
41.45
Characterization: Protein analysis
Protein Concentration Method
Not determined
Proteomics database
No
Characterization: Particle analysis
PMID previous EV particle analysis
Nanoparticle tracking analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
108
EV concentration
Yes
Particle yield
860000000000
EM
EM-type
Immune-EM
Proteïns
GFP
Image type
Close-up, Wide-field
Report size (nm)
60-200
Extra information
We have developed live cell imaging method to visualize and quantify exosome release (Verweij et al., JCB 2018). This method could be added to EV-track, e.g. as a measure to positively identify the endosomal origin of an EV population.
EV180082 10/10 Danio rerio Cell culture supernatant dUC Hyenne V 2019 57%

Study summary

Full title
All authors
Hyenne V, Ghoroghi S, Collot M, Bons J, Follain G, Harlepp S, Mary B, Bauer J, Mercier L, Busnelli I, Lefebvre O, Fekonja N, Garcia-Leon MJ, Machado P, Delalande F, López AA, Silva SG, Verweij FJ, van Niel G, Djouad F, Peinado H, Carapito C, Klymchenko AS, Goetz JG.
Journal
Dev cell
Abstract
Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly (show more...)Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo. (hide)
EV-METRIC
57% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Danio rerio
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Zmel1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
90
EV190080 1/3 Homo sapiens Cell culture supernatant DG
dUC
SEC
Zaborowski MP 2019 56%

Study summary

Full title
All authors
Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, Zappulli V, Maas SLN, Allen RM, Rumde P, György B, Aufiero M, Schweiger MW, Lai CP, Weissleder R, Lee H, Vickers KC, Tannous BA, Breakefield XO.
Journal
Sci Rep
Abstract
Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter (show more...)Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models. (hide)
EV-METRIC
56% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + SEC
Protein markers
EV: CD63
non-EV:
Proteomics
no
EV density (g/ml)
1.11
Show all info
Study aim
New methodological development/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
OVCAR5
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
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: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120101
Wash: volume per pellet (ml)
40
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
120101
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
8%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
MLS-50
Speed (g)
200620
Duration (min)
38
Fraction volume (mL)
350
Fraction processing
None
Size-exclusion chromatography
Total column volume (mL)
24
Sample volume/column (mL)
1
Resin type
Superdex 200
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63
Other 1
https://www.ncbi.nlm.nih.gov/pubmed/30943406
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
50-120
Other particle analysis name(1)
Report type
EV-concentration
EV190049 1/3 Homo sapiens Cell culture supernatant dUC
qEV
Bliss CM 2019 56%

Study summary

Full title
All authors
Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L.
Journal
Abstract
Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide)
EV-METRIC
56% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD81/ Alix/ CD63/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Antigen targeting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
NTA
Report type
Not Reported
EV190049 2/3 Homo sapiens Cell culture supernatant dUC
qEV
Bliss CM 2019 56%

Study summary

Full title
All authors
Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L.
Journal
Abstract
Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide)
EV-METRIC
56% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
Transfected with plasmid expressing EGFP
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD81/ Alix/ CD63/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Antigen targeting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Transfected with plasmid expressing EGFP
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
161
EV concentration
Yes
EV190049 3/3 Homo sapiens Cell culture supernatant dUC
qEV
Bliss CM 2019 56%

Study summary

Full title
All authors
Bliss CM, Parsons AJ, Nachbagauer R, Hamilton JR, Cappuccini F, Ulaszewska M, Webber JP, Clayton A, Hill AVS, Coughlan L.
Journal
Abstract
Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pr (show more...)Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5. (hide)
EV-METRIC
56% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
Transfected with plasmid expressing EGFP_C1C2
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD81/ Alix/ CD63/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Antigen targeting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Transfected with plasmid expressing EGFP_C1C2
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix
Not detected contaminants
GRP94
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
157
EV concentration
Yes
EV190027 1/1 Mus musculus Cell culture supernatant dUC Auber M 2019 56%

Study summary

Full title
All authors
Auber M, Fröhlich D, Drechsel O, Karaulanov E, Krämer-Albers EM.
Journal
J Extracell Vesicles
Abstract
Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from (show more...)Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from cultured cells may co-purify RNAs derived from media supplements such as fetal bovine serum (FBS) confounding EV-associated RNA. Defined culture media supplemented with a range of nutrient components provide an alternative to FBS addition and allow EV-collection under full medium conditions avoiding starvation and cell stress during the collection period. However, the potential contribution of serum-free media supplements to EV-RNA contamination has remained elusive and has never been assessed. Here, we report that RNA isolated from EVs harvested from cells under serum-replacement conditions includes miRNA contaminants carried into the sample by defined media components. Subjecting unconditioned, EV-free medium to differential centrifugation followed by reverse transcription quantitative PCR (RT-qPCR) on RNA isolated from the pellet resulted in detection of miRNAs that had been classified as EV-enriched by RNA-seq or RT-qPCR of an isolated EV-fraction. Ribonuclease (RNase-A) and detergent treatment removed most but not all of the contaminating miRNAs. Further analysis of the defined media constituents identified Catalase as a main source of miRNAs co-isolating together with EVs. Hence, miRNA contaminants can be carried into EV-samples even under serum-free harvesting conditions using culture media that are expected to be chemically defined. Formulation of miRNA-free media supplements may provide a solution to collect EVs clean from confounding miRNAs, which however still remains a challenging task. Differential analysis of EVs collected under full medium and supplement-deprived conditions appears to provide a strategy to discriminate confounding and EV-associated RNA. In conclusion, we recommend careful re-evaluation and validation of EV small RNA-seq and RT-qPCR datasets by determining potential medium background. (hide)
EV-METRIC
56% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary oligodendrocytes
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
103000
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Characterization: Particle analysis
NTA
Report type
Not Reported
EM
EV190026 1/2 Homo sapiens Serum dUC
qEV
Gualerzi A 2019 56%

Study summary

Full title
All authors
Gualerzi A, Picciolini S, Carlomagno C, Terenzi F, Ramat S, Sorbi S, Bedoni M.
Journal
Nanomedicine
Abstract
Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clin (show more...)Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clinical heterogeneity. Extracellular vesicles (EVs) were proposed as new biomarkers for PD because of their role as vehicles of multiple PD related molecules, but technical limitations exist in their detection and characterization in a clinical environment. We propose herein a Raman based protocol for the label-free analysis of circulating EVs as diagnostic and predictive tool for PD. After purification from serum of PD patients and healthy subjects, EVs were analyzed by Raman spectroscopy demonstrating the feasibility and reproducibility of the proposed biophotonic approach, its moderate accuracy in distinguishing PD patients from controls by their EV profile and the correlation between Raman data and clinical scales. Once validated, the Raman spectroscopy of circulating EVs could represent a reliable, automatable and sensitive method for the stratification of PD patients and for the evaluation of the effectiveness of rehabilitation and pharmacological treatments. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD63/ Flotillin1/ alpha-synuclein
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ alpha-synuclein
Detected contaminants
Albumin
EM
EM-type
Transmission-EM
Image type
Close-up
EV190026 2/2 Homo sapiens Serum dUC
qEV
Gualerzi A 2019 56%

Study summary

Full title
All authors
Gualerzi A, Picciolini S, Carlomagno C, Terenzi F, Ramat S, Sorbi S, Bedoni M.
Journal
Nanomedicine
Abstract
Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clin (show more...)Parkinson's disease (PD) is a chronic neurodegenerative disorder, characterized by considerable clinical heterogeneity. Extracellular vesicles (EVs) were proposed as new biomarkers for PD because of their role as vehicles of multiple PD related molecules, but technical limitations exist in their detection and characterization in a clinical environment. We propose herein a Raman based protocol for the label-free analysis of circulating EVs as diagnostic and predictive tool for PD. After purification from serum of PD patients and healthy subjects, EVs were analyzed by Raman spectroscopy demonstrating the feasibility and reproducibility of the proposed biophotonic approach, its moderate accuracy in distinguishing PD patients from controls by their EV profile and the correlation between Raman data and clinical scales. Once validated, the Raman spectroscopy of circulating EVs could represent a reliable, automatable and sensitive method for the stratification of PD patients and for the evaluation of the effectiveness of rehabilitation and pharmacological treatments. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Parkinson's disease
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + qEV
Protein markers
EV: CD63/ Flotillin1/ alpha-synuclein
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Parkinson's disease
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
100000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ alpha-synuclein
Detected contaminants
Albumin
EM
EM-type
Transmission-EM
Image type
Close-up
EV180082 6/10 Mus musculus Cell culture supernatant DG
dUC
Hyenne V 2019 56%

Study summary

Full title
All authors
Hyenne V, Ghoroghi S, Collot M, Bons J, Follain G, Harlepp S, Mary B, Bauer J, Mercier L, Busnelli I, Lefebvre O, Fekonja N, Garcia-Leon MJ, Machado P, Delalande F, López AA, Silva SG, Verweij FJ, van Niel G, Djouad F, Peinado H, Carapito C, Klymchenko AS, Goetz JG.
Journal
Dev cell
Abstract
Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly (show more...)Tumor extracellular vesicles (EVs) mediate the communication between tumor and stromal cells mostly to the benefit of tumor progression. Notably, tumor EVs travel in the bloodstream, reach distant organs, and locally modify the microenvironment. However, visualizing these events in vivo still faces major hurdles. Here, we describe an approach for tracking circulating tumor EVs in a living organism: we combine chemical and genetically encoded probes with the zebrafish embryo as an animal model. We provide a first description of tumor EVs hemodynamic behavior and document their intravascular arrest. We show that circulating tumor EVs are rapidly taken up by endothelial cells and blood patrolling macrophages and subsequently stored in degradative compartments. Finally, we demonstrate that tumor EVs activate macrophages and promote metastatic outgrowth. Overall, our study proves the usefulness and prospects of zebrafish embryo to track tumor EVs and dissect their role in metastatic niches formation in vivo. (hide)
EV-METRIC
56% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC
Protein markers
EV: Alix/ TSG101
non-EV:
Proteomics
yes
EV density (g/ml)
1.14
Show all info
Study aim
Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
4T1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
70
Wash: Rotor Type
SW 28
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 28
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
3
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ TSG101
Proteomics database
Yes
EV190040 3/12 Homo sapiens Cell culture supernatant DG
UF
Geeurickx E 2019 50%

Study summary

Full title
All authors
Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A.
Journal
Nat Commun
Abstract
Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
gag-EGFP
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + UF
Protein markers
EV:
non-EV:
Proteomics
yes
EV density (g/ml)
1.086-1.119
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
gag-EGFP
EV-producing cells
HEK293T
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
EV-subtype
Used subtypes
1.046 1.068 g/ml
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV190040 8/12 Homo sapiens Cell culture supernatant DG
UF
Geeurickx E 2019 50%

Study summary

Full title
All authors
Geeurickx E, Tulkens J, Dhondt B, Van Deun J, Lippens L, Vergauwen G, Heyrman E, De Sutter D, Gevaert K, Impens F, Miinalainen I, Van Bockstal PJ, De Beer T, Wauben MHM, Nolte-'t-Hoen ENM, Bloch K, Swinnen JV, van der Pol E, Nieuwland R, Braems G, Callewaert N, Mestdagh P, Vandesompele J, Denys H, Eyckerman S, De Wever O, Hendrix A.
Journal
Nat Commun
Abstract
Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological (show more...)Recent years have seen an increase of extracellular vesicle (EV) research geared towards biological understanding, diagnostics and therapy. However, EV data interpretation remains challenging owing to complexity of biofluids and technical variation introduced during sample preparation and analysis. To understand and mitigate these limitations, we generated trackable recombinant EV (rEV) as a biological reference material. Employing complementary characterization methods, we demonstrate that rEV are stable and bear physical and biochemical traits characteristic of sample EV. Furthermore, rEV can be quantified using fluorescence-, RNA- and protein-based technologies available in routine laboratories. Spiking rEV in biofluids allows recovery efficiencies of commonly implemented EV separation methods to be identified, intra-method and inter-user variability induced by sample handling to be defined, and to normalize and improve sensitivity of EV enumerations. We anticipate that rEV will aid EV-based sample preparation and analysis, data normalization, method development and instrument calibration in various research and biomedical applications. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + UF
Protein markers
EV:
non-EV:
Proteomics
yes
EV density (g/ml)
1.086-1.119
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary fibroblasts
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Proteomics database
Yes:
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV190003 1/1 Homo sapiens Urine dUC
PEG precipitation
dUC
Sabaratnam R 2019 50%

Study summary

Full title
All authors
Sabaratnam R, Geertsen L, Skjødt K, Hojlund K, Dimke H, Lund L, Svenningsen P.
Journal
Am J Physiol Renal Physiol
Abstract
Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumptio (show more...)Human urinary extracellular vesicles (uEVs) contain proteins from all nephron segments. An assumption for years has been that uEVs might provide a non-invasive liquid biopsy that reflect physiological regulation of transporter protein expression in human. We hypothesized that protein abundance in human kidney tissue and uEV are directly related and tested this in paired collections of nephrectomy tissue and urine sample from 12 patients. Kidney tissue was fractioned into total kidney protein, crude membrane (plasma membrane and large intracellular vesicles) and intracellular vesicle enriched fractions, as well as sections for immunolabelling. uEVs were isolated from spot urine samples. Antibodies were used to quantify 6 segment-specific proteins (proximal tubular expressed Na/Phosphate cotransporter NaPi-2a, thick ascending limb expressed Tamm-Horsfall protein and renal-outer-medullary K+channel ROMK, distal convoluted tubular expressed NaCl cotransporter NCC, intercalated cell expressed proton-pump subunit ATP6V1G3 and principal cell expressed aquaporin 2 (AQP2)) and 3 uEV markers (exosomal CD63, microvesicle marker VAMP3 and β-actin) in each fractions. By western blotting and immunofluorescence labelling, we found significant positive correlations between abundance of CD63, NCC, AQP2 and ATP6V1G3, respectively, within the different kidney-derived fractions. We detected all 9 proteins in uEVs, but their level did not correlate with kidney tissue protein abundance. The uEV protein levels showed higher inter-patient variability than the kidney-derived fractions, indicating that factors, besides kidney protein abundance, contribute to the uEV protein level. Our data suggest that, in a random sample of nephrectomy patients, uEV protein level is not a predictor of kidney protein abundance. (hide)
EV-METRIC
50% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
pre-nephrectomy
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC+ PEG precipitation + dUC
Protein markers
EV: SLC34A1/ VAMP3/ CD63/ beta-actin/ ROMK
non-EV: Tamm-Horsfall protein
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
pre-nephrectomy
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63/ VAMP3/ beta-actin/ SLC34A1/ ROMK
Detected contaminants
Tamm-Horsfall protein
EV180071 2/3 Homo sapiens Blood plasma SEC
UF
Brahmer A 2019 50%

Study summary

Full title
All authors
Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM.
Journal
J Extracell Vesicles
Abstract
Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide)
EV-METRIC
50% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
RQ 0.9 during exercise
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
SEC + UF
Protein markers
EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142
non-EV: / ApoA1
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
RQ 0.9 during exercise
Separation Method
Ultra filtration
Cut-off size (kDa)
30
Membrane type
Regenerated cellulose
Commercial kit
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD81/ TSG101/ CD9/ Syntenin/ CD41b/ CD63
Not detected EV-associated proteins
Sarcoglycan-alpha
Detected contaminants
ApoA1
Not detected contaminants
Detected EV-associated proteins
CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4
Characterization: Particle analysis
NTA
Report type
Not Reported
EV180071 3/3 Homo sapiens Blood plasma SEC
UF
Brahmer A 2019 50%

Study summary

Full title
All authors
Brahmer A, Neuberger E, Esch-Heisser L, Haller N, Jorgensen MM, Baek R, Möbius W, Simon P, Krämer-Albers EM.
Journal
J Extracell Vesicles
Abstract
Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physi (show more...)Physical activity initiates a wide range of multi-systemic adaptations that promote mental and physical health. Recent work demonstrated that exercise triggers the release of extracellular vesicles (EVs) into the circulation, possibly contributing to exercise-associated adaptive systemic signalling. Circulating EVs comprise a heterogeneous collection of different EV-subclasses released from various cell types. So far, a comprehensive picture of the parental and target cell types, EV-subpopulation diversity and functional properties of EVs released during exercise (ExerVs) is lacking. Here, we performed a detailed EV-phenotyping analysis to explore the cellular origin and potential subtypes of ExerVs. Healthy male athletes were subjected to an incremental cycling test until exhaustion and blood was drawn before, during, and immediately after the test. Analysis of total blood plasma by EV Array suggested endothelial and leukocyte characteristics of ExerVs. We further purified ExerVs from plasma by size exclusion chromatography as well as CD9-, CD63- or CD81-immunobead isolation to examine ExerV-subclass dynamics. EV-marker analysis demonstrated increasing EV-levels during cycling exercise, with highest levels at peak exercise in all EV-subclasses analysed. Phenotyping of ExerVs using a multiplexed flow-cytometry platform revealed a pattern of cell surface markers associated with ExerVs and identified lymphocytes (CD4, CD8), monocytes (CD14), platelets (CD41, CD42, CD62P), endothelial cells (CD105, CD146) and antigen presenting cells (MHC-II) as ExerV-parental cells. We conclude that multiple cell types associated with the circulatory system contribute to a pool of heterogeneous ExerVs, which may be involved in exercise-related signalling mechanisms and tissue crosstalk. (hide)
EV-METRIC
50% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
post exercise
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
SEC + UF
Protein markers
EV: TSG101/ CD31/ CD209/ CD326/ CD133/1/ CD8/ CD9/ CD49e/ CD81/ CD86/ Syntenin/ CD41b/ CD29/ CD63/ CD42a/ CD44/ CD20/ CD40/ Sarcoglycan-alpha/ CD24/ CD146/ CD69/ MHC2/ ROR1/ MHC1/ SSEA4/ CD105/ MCSP/ CD62p/ CD19/ CD142
non-EV: / ApoA1
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
post exercise
Separation Method
Ultra filtration
Cut-off size (kDa)
30
Membrane type
Regenerated cellulose
Commercial kit
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD81/ TSG101/ CD9/ Syntenin/ CD41b/ CD63
Not detected EV-associated proteins
Sarcoglycan-alpha
Detected contaminants
ApoA1
Not detected contaminants
Detected EV-associated proteins
CD63/ CD9/ CD81/ CD8/ CD19/ CD20/ CD24/ CD29/ CD31/ CD40/ CD41b/ CD42a/ CD44/ CD49e/ CD62p/ CD69/ CD86/ CD105/ CD133/1/ CD142/ CD146/ CD209/ CD326/ MHC1/ MHC2/ MCSP/ ROR1/ SSEA4
Characterization: Particle analysis
NTA
Report type
Not Reported
EV190092 2/2 Homo sapiens Cell culture supernatant dUC Brassart B 2019 44%

Study summary

Full title
All authors
Brassart B, Da Silva J, Donet M, Seurat E, Hague F, Terryn C, Velard F, Michel J, Ouadid-Ahidouch H, Monboisse JC, Hinek A, Maquart FX, Ramont L, Brassart-Pasco S.
Journal
Br J Cancer
Abstract
BACKGROUND: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elasto (show more...)BACKGROUND: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases. METHODS: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA. RESULTS: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding. CONCLUSIONS: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites. (hide)
EV-METRIC
44% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
elastin degradation product-stimulated
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD9/ CD81/ RhoA/ HSP90/ Actin/ Integrin-alphaV/ P-ERM/ MMP-2/ MMP-14
non-EV: GM130
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
elastin degradation product-stimulated
EV-producing cells
HT1080
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
JLA110
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Wash: time (min)
Wash: Rotor Type
Wash: speed (g)
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD9/ CD81/ RhoA/ HSP90/ Actin/ Integrin-alphaV/ P-ERM/ MMP-2/ MMP-14
Not detected contaminants
GM130
Fluorescent NTA
Relevant measurements variables specified?
NA
Detected EV-associated proteins
Not detected contaminants
Characterization: Particle analysis
NTA
Report type
EV concentration
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190011 2/5 Mus musculus Cell culture supernatant dUC Cianciaruso C 2019 44%

Study summary

Full title
All authors
Cianciaruso C, Beltraminelli T, Duval F, Nassiri S, Hamelin R, Mozes A, Gallart-Ayala H, Ceada Torres G, Torchia B, Ries CH, Ivanisevic J, De Palma M
Journal
Cell Rep
Abstract
Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor (show more...)Extracellular vesicles (EVs), including exosomes, modulate multiple aspects of cancer biology. Tumor-associated macrophages (TAMs) secrete EVs, but their molecular features and functions are poorly characterized. Here, we report methodology for the enrichment, quantification, and proteomic and lipidomic analysis of EVs released from mouse TAMs (TAM-EVs). Compared to source TAMs, TAM-EVs present molecular profiles associated with a Th1/M1 polarization signature, enhanced inflammation and immune response, and a more favorable patient prognosis. Accordingly, enriched TAM-EV preparations promote T cell proliferation and activation ex vivo. TAM-EVs also contain bioactive lipids and biosynthetic enzymes, which may alter pro-inflammatory signaling in the cancer cells. Thus, whereas TAMs are largely immunosuppressive, their EVs may have the potential to stimulate, rather than limit, anti-tumor immunity. (hide)
EV-METRIC
44% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD81/ Alix/ CD9/ GAPDH
non-EV:
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
E0771
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
35
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
134,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD9/ GAPDH
Not detected EV-associated proteins
CD81
EV170031 1/1 Homo sapiens Serum dUC
Filtration
Ramanathan S 2019 44%

Study summary

Full title
All authors
Ramanathan S, Douglas SR, Alexander GM, Shenoda BB, Barrett JE, Aradillas E, Sacan A, Ajit SK
Journal
J Transl Med
Abstract
BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employ (show more...)BACKGROUND: Therapeutic plasma exchange (PE) or plasmapheresis is an extracorporeal procedure employed to treat immunological disorders. Exosomes, nanosized vesicles of endosomal origin, mediate intercellular communication by transferring cargo proteins and nucleic acids and regulate many pathophysiological processes. Exosomal miRNAs are potential biomarkers due to their stability and dysregulation in diseases including complex regional pain syndrome (CRPS), a chronic pain disorder with persistent inflammation. A previous study showed that a subset of CRPS patients responded to PE. METHODS: As a proof-of-concept, we investigated the PE-induced exosomal miRNA changes in six CRPS patients. Plasma cytokine levels were measured by HPLC and correlated with miRNA expression. Luciferase assay following co-transfection of HEK293 cells with target 3'UTR constructs and miRNA mimics was used to evaluate miRNA mediated gene regulation of target mRNA. Transient transfection of THP-1 cells with miRNA mimics followed by estimation of target gene and protein expression was used to validate the findings. RESULTS: Comparison of miRNAs in exosomes from the serum of three responders and three poor-responders showed that 17 miRNAs differed significantly before and after therapy. Of these, poor responders had lower exosomal hsa-miR-338-5p. We show that miR-338-5p can bind to the interleukin 6 (IL-6) 3' untranslated region and can regulate IL-6 mRNA and protein levels in vitro. PE resulted in a significant reduction of IL-6 in CRPS patients. CONCLUSIONS: We propose that lower pretreatment levels of miR-338-5p in poor responders are linked to IL-6 levels and inflammation in CRPS. Our data suggests the feasibility of exploring exosomal miRNAs as a strategy in patient stratification for maximizing therapeutic outcome of PE. (hide)
EV-METRIC
44% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Complex regional pain syndrome
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Filtration
Adj. k-factor
142.9 (pelleting) / 142.9 (washing)
Protein markers
EV: CD81/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Complex regional pain syndrome
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
142.9
Wash: time (min)
70
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
110000
Wash: adjusted k-factor
142.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
85.7
EV concentration
Yes
Particle yield
394000000000
EM
EM-type
Transmission-EM/ Immune-EM
Proteïns
CD81
Image type
Close-up, Wide-field
EV190020 3/3 Rattus norvegicus Cell culture supernatant DG
dUC
Filtration
Kyuno, Daisuke 2019 43%

Study summary

Full title
All authors
Kyuno D, Zhao K, Schnölzer M, Provaznik J, Hackert T, Zöller M.
Journal
Int J Cancer
Abstract
Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdow (show more...)Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor-exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEX were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX. This article is protected by copyright. All rights reserved. (hide)
EV-METRIC
43% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
claudin 7 knockdown
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Protein markers
EV:
non-EV:
Proteomics
no
EV density (g/ml)
1.15-1.56
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
claudin 7 knockdown
EV-producing cells
ASML
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
120
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
4
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
1.28
Fraction processing
Centrifugation
Pelleting: volume per fraction
50
Pelleting: duration (min)
150
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
50
Pelleting-wash: duration (min)
150
Pelleting-wash: speed (g)
Type 45 Ti
Filtration steps
0.22µm or 0.2µm
Protein Concentration Method
Bradford
EV170033 2/2 Homo sapiens Blood plasma SEC Roura S 2019 42%

Study summary

Full title
All authors
Roura S, Gámez-Valero A, Lupón J, Gálvez-Montón C, Borràs FE, Bayes-Genis A.
Journal
Lab Invest.
Abstract
Dilated cardiomyopathy (DCM) remains a major cause of heart failure and carries a poor prognosis des (show more...)Dilated cardiomyopathy (DCM) remains a major cause of heart failure and carries a poor prognosis despite important advances in recent years. Better disease characterization using novel molecular techniques is needed to refine its progression. This study explored the proteomic signature of plasma-derived extracellular vesicles (EVs) obtained from DCM patients and healthy controls using size-exclusion chromatography (SEC). EV-enriched fractions were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Raw data obtained from LC-MS/MS were analyzed against the Uniprot human database using MaxQuant software. Additional analyses using Perseus software were based on the Intensity-Based Absolute Quantification (iBAQ) values from MaxQuan