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You searched for: EV220024 (EV-TRACK ID)

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Results list Study Tree
Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV220024 1/7 Homo sapiens MDA-MB-231 DG
Filtration
UF
SEC (non-commercial) 		Roux, Quentin 2023 100%

Study summary

Full title
Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
EV density (g/ml)
1.09-1.11
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
180000000
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
0.8
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ FGF-2/ Fractalkine/ IL-1RA/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 2/7 Homo sapiens MCF-7 DG
Filtration
UF
SEC (non-commercial) 		Roux, Quentin 2023 100%

Study summary

Full title
Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
EV density (g/ml)
1.09-1.11
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MCF-7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
179999999
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
0.8
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 4/7 Homo sapiens Immortalized patient-derived breast CAF DG
Filtration
UF
SEC (non-commercial) 		Roux, Quentin 2023 100%

Study summary

Full title
Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
EV density (g/ml)
1.09-1.11
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Immortalized patient-derived breast CAF
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Cell count
120000000
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
0.8
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 3/7 Homo sapiens Immortalized patient-derived breast CAF (d)(U)C Roux, Quentin 2023 78%

Study summary

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

Study summary

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

Study summary

Full title
Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: Argonaute 2
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
180000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
60
Wash: Rotor Type
SW 32.1 Ti
Wash: speed (g)
100000
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Detected contaminants
Argonaute 2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 7/7 Homo sapiens MDA-MB-231 (d)(U)C
SEC (non-commercial) 		Roux, Quentin 2023 33%

Study summary

Full title
Depletion of soluble cytokines unlocks the immunomodulatory bioactivity of extracellular vesicles
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
33% (76th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDA-MB-231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
95
Cell count
180000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
60
Wash: Rotor Type
SW 32.1 Ti
Wash: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
None
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
1 - 7 of 7
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV220024
species
Homo
sapiens
sample type
Cell
culture
cell type
MDA-MB-231
MCF-7
Immortalized
patient-derived
breast
CAF
Immortalized
patient-derived
breast
CAF
Immortalized
patient-derived
breast
CAF
MDA-MB-231
MDA-MB-231
medium
EV-depleted
medium
EV-depleted
medium
Serum
free
medium
Serum
free
medium
Serum
free
medium
EV-depleted
medium
EV-depleted
medium
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
separation protocol
Density
gradient/
Filtration/
Ultrafiltration/
Size-exclusion
chromatography
(non-commercial)
Density
gradient/
Filtration/
Ultrafiltration/
Size-exclusion
chromatography
(non-commercial)
Density
gradient/
Filtration/
Ultrafiltration/
Size-exclusion
chromatography
(non-commercial)
dUC
Filtration/
Ultrafiltration/
Size-exclusion
chromatography
(non-commercial)
dUC
dUC/
Size-exclusion
chromatography
(non-commercial)
Exp. nr.
1
2
4
3
5
6
7
EV-METRIC %
100
100
100
78
78
78
33