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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV220304 6/30 Homo sapiens Blood plasma DG
UF
SEC (non-commercial)
Dhondt B 2023 100%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (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
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD81/ Flotillin-1
non-EV: ApoA1/ Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD81/ Flotillin-1
Detected contaminants
ApoA1
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220024 1/7 Homo sapiens MDA-MB-231 DG
Filtration
UF
SEC (non-commercial)
Roux, Quentin 2023 100%

Study summary

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

Study summary

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

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
EV density (g/ml)
1.09-1.11
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Immortalized patient-derived breast CAF
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Cell count
120000000
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.8
Sample volume (mL)
0.8
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV230008 4/42 Mus musculus EO771 (d)(U)C
DG
UF
Cocozza F 2023 89%

Study summary

Full title
All authors
Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C
Journal
EMBO J
Abstract
Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
sEV
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Ultrafiltration
Protein markers
EV: Alix/ CD9/ CD63/ HSP90/ MHC1/ MFGE8
non-EV: Argonaute-2
Proteomics
yes
EV density (g/ml)
1.015-1.085
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
EO771
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Cell count
100000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
200000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
6%
Highest density fraction
22%
Total gradient volume, incl. sample (mL)
16
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
187000
Duration (min)
90
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
6
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
2.29E
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.015-1.035
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ HSP90/ MHC1/ MFGE8
Detected contaminants
Argonaute-2
Proteomics database
PRIDE
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
135
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Wide-field
EV230008 5/42 Mus musculus EO771 (d)(U)C
DG
UF
Cocozza F 2023 89%

Study summary

Full title
All authors
Cocozza F, Martin-Jaular L, Lippens L, Di Cicco A, Arribas YA, Ansart N, Dingli F, Richard M, Merle L, Jouve San Roman M, Poullet P, Loew D, Lévy D, Hendrix A, Kassiotis G, Joliot A, Tkach M, Théry C
Journal
EMBO J
Abstract
Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or (show more...)Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
VLP
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Ultrafiltration
Protein markers
EV: Alix/ CD9/ CD63/ HSP90/ MHC1/ MFGE8
non-EV: Argonaute-2
Proteomics
yes
EV density (g/ml)
1.015-1.085
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
EO771
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Cell count
100000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
200000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
6%
Highest density fraction
22%
Total gradient volume, incl. sample (mL)
16
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
187000
Duration (min)
90
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
6
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
2.29E
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
1.065-1.085
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ HSP90/ MHC1/ MFGE8
Not detected contaminants
Argonaute-2
Proteomics database
PRIDE
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
135
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Wide-field
EV220090 1/6 Homo sapiens wound fluid (d)(U)C
Filtration
IAF
UF
DG
Guda PR 2023 89%

Study summary

Full title
All authors
Guda PR, Sharma A, Anthony AJ, ElMasry MS, Couse AD, Ghatak PD, Das A, Timsina L, Trinidad JC, Roy S, Clemmer DE, Sen CK, Ghatak S
Journal
Nano Today
Abstract
Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine s (show more...)Exosomes, a class of extracellular vesicles of endocytic origin, play a critical role in paracrine signaling for successful cell-cell crosstalk . However, limitations in our current understanding of these circulating nanoparticles hinder efficient isolation, characterization, and downstream functional analysis of cell-specific exosomes. In this work, we sought to develop a method to isolate and characterize keratinocyte-originated exosomes () from human chronic wound fluid. Furthermore, we studied the significance of in diabetic wounds. LC-MS-MS detection of KRT14 in and subsequent validation by Vesiclepedia and Exocarta databases identified surface KRT14 as a reliable marker of . dSTORM nanoimaging identified KRT14 extracellular vesicles () in human chronic wound fluid, 23% of which were of exosomal origin. An immunomagnetic two-step separation method using KRT14 and tetraspanin antibodies successfully isolated from the heterogeneous pool of EV in chronic wound fluid of 15 non-diabetic and 22 diabetic patients. Isolated (Ø75-150nm) were characterized per EV-track guidelines. dSTORM images, analyzed using online CODI followed by independent validation using Nanometrix, revealed Ø as 80-145nm. The abundance of was low in diabetic wound fluids and negatively correlated with patient HbA1c levels. The isolated from diabetic wound fluid showed a low abundance of small bp RNA (<200 bp). Raman spectroscopy underscored differences in surface lipids between non-diabetic and diabetic Uptake of by monocyte-derived macrophages (MDM) was low for diabetics non-diabetics. Unlike from non-diabetics, the addition of diabetic to MDM polarized with LPS and INFγ resulted in sustained expression of iNOS and pro-inflammatory chemokines known to recruit macrophage (mϕ) This work provides maiden insight into the structure, composition, and function of from chronic wound fluid thus providing a foundation for the study of exosomal malfunction under conditions of diabetic complications such as wound chronicity. (hide)
EV-METRIC
89% (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. 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
wound fluid
Sample origin
Non-diabetic
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Immunoaffinity capture (non-commercial)
Ultrafiltration
Density gradient
Protein markers
EV: HSP90/ Alix/ CD81/ Flotillin-1/ TSG101/ ANXA5/ ICAM/ CD63
non-EV: Prohibitin/ GM130
Proteomics
yes
EV density (g/ml)
1.15-1.2
Show all info
Study aim
Function/Biomarker/Mechanism of uptake/transfer/New methodological development/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
wound fluid
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
TLA-120.2
Pelleting: speed (g)
245000
Wash: volume per pellet (ml)
0.5
Wash: time (min)
120
Wash: Rotor Type
TLA-120.2
Wash: speed (g)
245000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
0.8M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
0.9
Sample volume (mL)
0.15
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.15
Fraction processing
Ultracentrifugation
Pelleting: volume per fraction
0.5
Pelleting: duration (min)
120
Pelleting: rotor type
TLA-120.2
Pelleting: speed (g)
245000
Filtration steps
0.2 or 0.22 µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Immunoaffinity capture
Selected surface protein(s)
CD9/CD63/CD81/ KRT14
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per E08 particles: 0.55
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
HSP90
Not detected contaminants
Prohibitin
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
Yes
Selected surface protein(s)
CD9/ CD63/ CD81/ KRT14
Proteomics database
No
Detected EV-associated proteins
Alix/ CD81/ Flotillin-1/ TSG101/ ANXA5/ ICAM
Not detected EV-associated proteins
CD63/ EPCAM
Detected contaminants
GM130
Detected EV-associated proteins
CD9/C63/CD81/ KRT5
Characterization: RNA analysis
RNA analysis
Type
Capillary electrophoresis (e.g. Bioanalyzer)
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
87
EV concentration
Yes
Particle yield
as particles per mg of albumin: 2.30E+08
EM
EM-type
Scanning-EM/ Immuno-EM
EM protein
TSG101
Image type
Close-up, Wide-field
EV210141 2/5 Homo sapiens human umbilical vein endothelial cells IAF
Ultrafiltratrion
(d)(U)C
DG
Zhao F 2023 89%

Study summary

Full title
All authors
Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G
Journal
EMBO J
Abstract
Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
ZIKV infected cells
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Immunoaffinity capture (non-commercial)
Ultrafiltratrion
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9
non-EV: Capsid/ E/ LC3/ Calnexin
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human umbilical vein endothelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
2,00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0%
Highest density fraction
80%
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
P55ST
Speed (g)
250000
Duration (min)
1080
Fraction volume (mL)
0,3
Ultra filtration
Cut-off size (kDa)
100 kDa
Membrane type
Regenerated cellulose
Immunoaffinity capture
Selected surface protein(s)
CD9
Other
Name other separation method
Ultrafiltratrion
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81
Detected contaminants
Capsid/ E
Not detected contaminants
LC3/ Calnexin
Characterization: RNA analysis
RNA analysis
Type
RT(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Wide-field
Report size (nm)
100
EV210141 3/5 Homo sapiens human umbilical vein endothelial cells Ultrafiltratrion
(d)(U)C
DG
Zhao F 2023 89%

Study summary

Full title
All authors
Zhao F, Xu Y, Liu N, Lv D, Chen Y, Liu Z, Jin X, Xiao M, Lavillette D, Zhong J, Bartenschlager R, Long G
Journal
EMBO J
Abstract
Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some pa (show more...)Mosquito-borne flaviviruses including Zika virus (ZIKV) represent a public health problem in some parts of the world. Although ZIKV infection is predominantly asymptomatic or associated with mild symptoms, it can lead to neurological complications. ZIKV infection can also cause antibody-dependent enhancement (ADE) of infection with similar viruses, warranting further studies of virion assembly and the function of envelope (E) protein-specific antibodies. Although extracellular vesicles (EVs) from flavivirus-infected cells have been reported to transmit infection, this interpretation is challenged by difficulties in separating EVs from flavivirions due to their similar biochemical composition and biophysical properties. In the present study, a rigorous EV-virion separation method combining sequential ultracentrifugation and affinity capture was developed to study EVs from ZIKV-infected cells. We find that these EVs do not transmit infection, but EVs display abundant E proteins which have an antigenic landscape similar to that of virions carrying E. ZIKV E-coated EVs attenuate antibody-dependent enhancement mediated by ZIKV E-specific and DENV-cross-reactive antibodies in both cell culture and mouse models. We thus report an alternative route for Flavivirus E protein secretion. These results suggest that modulation of E protein release via virions and EVs may present a new approach to regulating flavivirus-host interactions. (hide)
EV-METRIC
89% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
ZIKV infected cells
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltratrion
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: TSG101/ CD63/ CD81/ Alix/ HSP70/ CD9
non-EV: Capsid/ E/ LC3/ Calnexin
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
human umbilical vein endothelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
2,00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0%
Highest density fraction
80%
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
P55ST
Speed (g)
250000
Duration (min)
1080
Fraction volume (mL)
0,3
Ultra filtration
Cut-off size (kDa)
100 kDa
Membrane type
Regenerated cellulose
Other
Name other separation method
Ultrafiltratrion
Characterization: Protein analysis
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ TSG101/ HSP70/ Alix/ CD81
Detected contaminants
Capsid/ E
Not detected contaminants
LC3/ Calnexin
Characterization: RNA analysis
RNA analysis
Type
RT(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
100
EV210215 3/4 Homo sapiens PBS spiked with recombinant EV (gag-EGFP HEK293T) DG Van Dorpe S 2023 88%

Study summary

Full title
All authors
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A
Journal
J Nanobiotechnology
Abstract
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide)
EV-METRIC
88% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
PBS spiked with recombinant EV (gag-EGFP HEK293T)
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Protein markers
EV: TSG101/ CD81/ Alix/ p24/ CD9/ syntenin-1
non-EV: None
Proteomics
no
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
PBS spiked with recombinant EV (gag-EGFP HEK293T)
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Concentration Method
Not determined
Protein Yield (µg)
as percentage of spiked rEV
ELISA
Antibody details provided?
No
Detected EV-associated proteins
p24
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
140
EV concentration
Yes
Particle yield
as percentage of spiked rEV
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Not reported
EV210215 4/4 Homo sapiens PBS spiked with recombinant EV (gag-EGFP HEK293T) DG Van Dorpe S 2023 88%

Study summary

Full title
All authors
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A
Journal
J Nanobiotechnology
Abstract
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide)
EV-METRIC
88% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
PBS spiked with recombinant EV (gag-EGFP HEK293T)
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Protein markers
EV: TSG101/ CD81/ Alix/ p24/ CD9/ syntenin-1
non-EV: None
Proteomics
no
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
PBS spiked with recombinant EV (gag-EGFP HEK293T)
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.8
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Not determined
ELISA
Antibody details provided?
No
Detected EV-associated proteins
p24
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
140
EV concentration
Yes
Particle yield
as percentage of spiked rEV
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Not reported
EV210215 1/4 Homo sapiens Blood plasma (d)(U)C
DG
UF
SEC (non-commercial)
Van Dorpe S 2023 86%

Study summary

Full title
All authors
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A
Journal
J Nanobiotechnology
Abstract
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide)
EV-METRIC
86% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Breast cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Albumin/ ApoA1/ ApoB
Proteomics
yes
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange Consortium
Detected contaminants
Albumin/ ApoA1/ ApoB
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV210215 2/4 Homo sapiens urine (d)(U)C
DG
UF
Van Dorpe S 2023 86%

Study summary

Full title
All authors
Van Dorpe S, Lippens L, Boiy R, Pinheiro C, Vergauwen G, Rappu P, Miinalainen I, Tummers P, Denys H, De Wever O, Hendrix A
Journal
J Nanobiotechnology
Abstract
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for (show more...)Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers. (hide)
EV-METRIC
86% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Ultrafiltration
Protein markers
EV: None
non-EV: Albumin/ Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.086-1.119
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
urine
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Bottom-up
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange Consortium
Detected contaminants
Albumin/ Tamm-Horsfall protein
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV220304 3/30 Homo sapiens Blood plasma DG
UF
SEC (non-commercial)
Dhondt B 2023 83%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide)
EV-METRIC
83% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ Calreticulin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220304 9/30 Homo sapiens Blood plasma DG
UF
SEC (non-commercial)
Dhondt B 2023 83%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide)
EV-METRIC
83% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220304 12/30 Homo sapiens Serum DG
UF
SEC (non-commercial)
Dhondt B 2023 83%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide)
EV-METRIC
83% (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
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220304 15/30 NA NA DG
UF
SEC (non-commercial)
Dhondt B 2023 83%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide)
EV-METRIC
83% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
NA
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
NA
Sample Type
NA
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220304 18/30 NA NA DG
UF
SEC (non-commercial)
Dhondt B 2023 83%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide)
EV-METRIC
83% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
NA
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
NA
Sample Type
NA
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220304 21/30 NA NA DG
UF
SEC (non-commercial)
Dhondt B 2023 83%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide)
EV-METRIC
83% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
NA
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
NA
Sample Type
NA
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220304 24/30 NA NA DG
UF
SEC (non-commercial)
Dhondt B 2023 83%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide)
EV-METRIC
83% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
NA
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
NA
Sample Type
NA
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220304 30/30 NA NA DG
UF
SEC (non-commercial)
Dhondt B 2023 83%

Study summary

Full title
All authors
Dhondt B, Pinheiro C, Geeurickx E, Tulkens J, Vergauwen G, Van Der Pol E, Nieuwland R, Decock A, Miinalainen I, Rappu P, Schroth G, Kuersten S, Vandesompele J, Mestdagh P, Lumen N, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and hold (show more...)The analysis of extracellular vesicles (EV) in blood samples is under intense investigation and holds the potential to deliver clinically meaningful biomarkers for health and disease. Technical variation must be minimized to confidently assess EV-associated biomarkers, but the impact of pre-analytics on EV characteristics in blood samples remains minimally explored. We present the results from the first large-scale EV Blood Benchmarking (EVBB) study in which we systematically compared 11 blood collection tubes (BCT/ six preservation and five non-preservation) and three blood processing intervals (BPI/ 1, 8 and 72 h) on defined performance metrics (n = 9). The EVBB study identifies a significant impact of multiple BCT and BPI on a diverse set of metrics reflecting blood sample quality, ex-vivo generation of blood-cell derived EV, EV recovery and EV-associated molecular signatures. The results assist the informed selection of the optimal BCT and BPI for EV analysis. The proposed metrics serve as a framework to guide future research on pre-analytics and further support methodological standardization of EV studies. (hide)
EV-METRIC
83% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
NA
Sample origin
NA
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins/ Albumin/ GM130/ PMP70/ Prohibitin
Proteomics
yes
EV density (g/ml)
1.09-1.10
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV--related methods
Sample
Species
NA
Sample Type
NA
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Proteomics database
ProteomeXchange
Detected contaminants
Argonaute-2/ Calreticulin/ Complement factors/ Immunoglobulins/ Apolipoproteins
Not detected contaminants
Albumin/ GM130/ PMP70/ Prohibitin
Characterization: RNA analysis
RNA analysis
Type
RNA -sequencing
Database
BioProject
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-250
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220429 1/1 Mus musculus Osteoclasts (d)(U)C Faqeer, Abdullah 2023 78%

Study summary

Full title
All authors
Abdullah Faqeer, Mengzhen Wang, Gulzar Alam, Arshad Ahmed Padhiar, Dexiu Zheng, Zhiming Luo, Irene Shuping Zhao, Guangqian Zhou, Jeroen van den Beucken, Huanan Wang, Yang Zhang
Journal
Biomaterials
Abstract
Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbi (show more...)Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) to maintain bone architecture and systemic mineral homeostasis throughout life. However, the mechanisms responsible for the coupling between OCs and OBs have not been fully elucidated. Herein, we first validate that secreted extracellular vesicles by osteoclasts (OC-EVs) promote osteogenic differentiation of mesenchymal stem cells (MSCs) and further demonstrate the efficacy of osteoclasts and their secreted EVs in treating tibial bone defects. Furthermore, we show that OC-EVs contain several osteogenesis-promoting proteins as cargo. By employing proteomic and functional analysis, we reveal that mature osteoclasts secrete thrombin cleaved phosphoprotein 1 (SPP1) through extracellular vesicles which triggers MSCs osteogenic differentiation into OBs by activating Transforming Growth Factor β1 (TGFβ1) and Smad family member 3 (SMAD3) signaling. In conclusion, our findings prove an important role of SPP1, present as cargo in OC-derived EVs, in signaling to MSCs and driving their differentiation into OBs. This biological mechanism implies a paradigm shift regarding the role of osteoclasts and their signaling toward the treatment of skeletal disorders which require bone formation. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: HSPA8/ CD9/ CD81/ TSG101/ beta actin
non-EV: calnexin
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
Osteoclasts
EV-harvesting Medium
Serum free medium
Cell count
3000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70.1Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
8
Wash: time (min)
60
Wash: Rotor Type
Type 70.1Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
HSPA8/ CD9/ CD81/ TSG101/ beta actin
Not detected contaminants
calnexin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
180
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
155
EV220369 1/5 Homo sapiens Blood plasma (d)(U)C Lapin M 2023 78%

Study summary

Full title
All authors
Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O
Journal
PLoS One
Abstract
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD9/ CD63/ CD81/ TSG101
non-EV: ApoA1
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
20
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ TSG101
Detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
1-10000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Equal or larger than 200nm
EV220369 2/5 Homo sapiens Blood plasma (d)(U)C
Filtration
qEV
UF
Lapin M 2023 78%

Study summary

Full title
All authors
Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O
Journal
PLoS One
Abstract
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Commercial method
Ultrafiltration
Protein markers
EV: CD9/ CD63/ CD81/ TSG101
non-EV: ApoA1
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Larger than 0.45 µm
Ultra filtration
Cut-off size (kDa)
100 kDa
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ TSG101
Detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
1-10000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Below or equal to 200nm
EV220369 3/5 Homo sapiens Blood plasma (d)(U)C
Filtration
ExoEasy
Lapin M 2023 78%

Study summary

Full title
All authors
Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O
Journal
PLoS One
Abstract
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Commercial method
Protein markers
EV: CD9/ CD63/ CD81/ TSG101
non-EV: ApoA1
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
Larger than 0.45 µm
Commercial kit
ExoEasy
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ TSG101
Detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
1-10000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Below or equal to 200nm
EV220369 5/5 Homo sapiens Blood plasma (d)(U)C
Filtration
Lapin M 2023 78%

Study summary

Full title
All authors
Lapin M, Tjensvoll K, Nedrebø K, Taksdal E, Janssen H, Gilje B, Nordgård O
Journal
PLoS One
Abstract
Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Sev (show more...)Tumor-derived extracellular vesicles (EVs) are reported to contain nucleic acids, including DNA. Several studies have highlighted the potential of EV-derived DNA (evDNA) as a circulating biomarker, even demonstrating that evDNA can outperform cell-free DNA (cfDNA) in terms of sensitivity. Here, we evaluated EVs as a potential source of tumor-derived DNA in patients with advanced pancreatic cancer. evDNA from both DNase-treated and untreated EV samples was analyzed to determine whether the DNA was primarily located internally or outside (surface-bound) the EVs. To assess whether methodology affected the results, we isolated EVs using four different methods for small EV isolation and differential centrifugation for isolating large EVs. Our results indicated that the DNA content of EVs was significantly less than the cfDNA content isolated from the same plasma volume (p < 0.001). Most of the detected evDNA was also located on the outside of the vesicles. Furthermore, the fraction of tumor-derived DNA in EVs was similar to that found in cfDNA. In conclusion, our results suggest that quantification of evDNA, as a source of tumor-derived DNA, does not add information to that obtained with cfDNA, at least not in patients with advanced pancreatic cancer. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Pancreatic Cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
32
Wash: time (min)
70
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100,000
Filtration steps
Larger than 0.45 µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81/ TSG101
Detected contaminants
ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
1-10000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
Below or equal to 200nm
EV220024 3/7 Homo sapiens Immortalized patient-derived breast CAF (d)(U)C Roux, Quentin 2023 78%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Immortalized patient-derived breast CAF
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Cell count
120000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
16
Wash: time (min)
60
Wash: Rotor Type
SW 32.1 Ti
Wash: speed (g)
100000
Size-exclusion chromatography
Resin type
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ TSG101
Detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
VEGF-A/ MCP-1/ FGF-2/ Fractalkine/ IL-1RA/ PDGF-AA/ IL-8/ GRO_
Not detected EV-associated proteins
sCD40L/ EGF/ Eotaxin/ Flt-3L/ G-CSF/ GM-CSF/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PD
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 5/7 Homo sapiens Immortalized patient-derived breast CAF Filtration
UF
SEC (non-commercial)
Roux, Quentin 2023 78%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ CD9/ TSG101/ sCD40L/ EGF/ Eotaxin/ FGF-2/ Flt-3L/ Fractalkine/ G-CSF/ GM-CSF/ GRO_/ IFN_2/ IFN_/ IL-1_/ IL-1_/ IL-1RA/ IL-2/ IL-3/ IL-4/ IL-5/ IL-6/ IL-7/ IL-8/ IL-9/ IL-10/ IL-12p40/ IL-12p70/ IL-13/ IL-15/ IL-17A/ IP-10/ MCP-1/ MCP-3/ MDC/ MIP-1_/ MIP-1_/ PDGF-AA/ PDGF-BB/ RANTES/ TGF_/ TNF_
non-EV: Argonaute 2
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Immortalized patient-derived breast CAF
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Cell count
120000000
Separation Method
Filtration steps
0.45 µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ TSG101
Not detected contaminants
Argonaute 2
Other 1
Luminex
Detected EV-associated proteins
to complete
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EV concentration
Yes
Particle yield
per milliliter of starting sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-100
EV220024 6/7 Homo sapiens MDA-MB-231 (d)(U)C Roux, Quentin 2023 78%

Study summary

Full title
All authors
Quentin Roux, Robin Boiy, Felix De Vuyst, Mercedes Tkach, Claudio Pinheiro, Sofie de Geyter, Ilkka Miinalainen, Clotilde Théry, Olivier De Wever, An Hendrix
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in phys (show more...)Despite an enormous interest in understanding the bioactivity of extracellular vesicles (EV) in physiology and disease for the development of therapeutic applications, the impact of EV preparation methods remains minimally explored. In this study, we implemented density gradient ultracentrifugation combined with size-exclusion chromatography (DG-SEC), differential ultracentrifugation (dUC) and/or stand-alone SEC (sSEC) to fractionate media conditioned by different cancer cells and/or cancer-associated fibroblasts (CAF). EV-enriched but protein-depleted versus EV-depleted but protein-enriched DG-SEC fractions, and EV-containing dUC and sSEC preparations were quality controlled for particle number, protein concentration, selected protein composition and ultrastructure, characterized for their cytokine content, and dose-dependently evaluated for monocyte-derived dendritic cell (MoDC) maturation by measuring surface marker expression and/or cytokine secretion. EV preparations obtained by DG-SEC from media conditioned by different cancer cell lines or CAF, were depleted from soluble immune suppressive cytokines such as VEGF-A and MCP-1 and potently stimulated MoDC maturation. In contrast, EV-containing dUC or sSEC preparations were not depleted from these soluble cytokines and were unable to mature MoDC. Subsequent processing of dUC EV preparations by SEC dose-dependently restored the immunomodulatory bioactivity. Overall, our results demonstrate that method-dependent off-target enrichment of soluble cytokines has implications for the study of EV immunomodulatory bioactivity and warrants careful consideration. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
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
EV210382 1/4 Homo sapiens maternal placenta perfusate (d)(U)C Awoyemi T 2023 78%

Study summary

Full title
All authors
Awoyemi T, Zhang W, Rahbar M, Cribbs A, Logenthiran P, Jiang S, Collett G, Cerdeira AS, Vatish M
Journal
Front Cardiovasc Med
Abstract
Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies world (show more...)Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
maternal placenta perfusate
Sample origin
Control condition
Focus vesicles
medium/large 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/ PLAP/ CD9
non-EV: Cytochrome C
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
maternal placenta perfusate
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
30
Pelleting: rotor type
Sorvall TST28.39
Pelleting: speed (g)
10,000
Wash: volume per pellet (ml)
45
Wash: time (min)
30
Wash: Rotor Type
Sorvall TST28.39
Wash: speed (g)
10,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
8680-10240
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ PLAP
Not detected EV-associated proteins
CD9
Not detected contaminants
Cytochrome C
Proteomics database
PRIDE
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer)
Database
NCBI geo
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
205.8
EV concentration
Yes
Particle yield
6.98E11-1.12E12
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210382 2/4 Homo sapiens maternal placenta perfusate (d)(U)C Awoyemi T 2023 78%

Study summary

Full title
All authors
Awoyemi T, Zhang W, Rahbar M, Cribbs A, Logenthiran P, Jiang S, Collett G, Cerdeira AS, Vatish M
Journal
Front Cardiovasc Med
Abstract
Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies world (show more...)Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
maternal placenta perfusate
Sample origin
Control condition
Focus vesicles
small extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD63/ PLAP/ CD9
non-EV: Cytochrome C
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
maternal placenta perfusate
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Sorvall TST28.39
Pelleting: speed (g)
150,000
Wash: volume per pellet (ml)
45
Wash: time (min)
120
Wash: Rotor Type
Sorvall TST28.39
Wash: speed (g)
150,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
2250-3040
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD63/ PLAP
Not detected EV-associated proteins
CD9
Not detected contaminants
Cytochrome C
Flow cytometry
Type of Flow cytometry
A BD LSRII flow cytometer (BD Biosciences) with a blue, violet, and red laser
Calibration bead size
0.11/ 0.18/ 0.24/ 0.30/ 0.50/ 0.59/ 0.88/ 1.30
Antibody details provided?
No
Proteomics database
PRIDE
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer)
Database
NCBI geo
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
479
EV concentration
Yes
Particle yield
1.59E11-1.78E11
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210382 3/4 Homo sapiens maternal placenta perfusate (d)(U)C Awoyemi T 2023 78%

Study summary

Full title
All authors
Awoyemi T, Zhang W, Rahbar M, Cribbs A, Logenthiran P, Jiang S, Collett G, Cerdeira AS, Vatish M
Journal
Front Cardiovasc Med
Abstract
Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies world (show more...)Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
maternal placenta perfusate
Sample origin
Preeclampsia
Focus vesicles
medium/large extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ PLAP/ CD9
non-EV: Cytochrome C
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
maternal placenta perfusate
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
Yes
Pelleting: time(min)
30
Pelleting: rotor type
Sorvall TST28.39
Pelleting: speed (g)
10,000
Wash: volume per pellet (ml)
45
Wash: time (min)
30
Wash: Rotor Type
Sorvall TST28.39
Wash: speed (g)
10,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
10460-21650
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ PLAP
Not detected EV-associated proteins
CD9
Not detected contaminants
Cytochrome C
Proteomics database
PRIDE
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer)
Database
NCBI geo
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
205.8
EV concentration
Yes
Particle yield
3.75E11-1E12
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210382 4/4 Homo sapiens maternal placenta perfusate (d)(U)C Awoyemi T 2023 78%

Study summary

Full title
All authors
Awoyemi T, Zhang W, Rahbar M, Cribbs A, Logenthiran P, Jiang S, Collett G, Cerdeira AS, Vatish M
Journal
Front Cardiovasc Med
Abstract
Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies world (show more...)Preeclampsia (PE) is a pregnancy-specific hypertensive disorder affecting 2%-8% of pregnancies worldwide. Biomarker(s) for the disorder exists, but while these have excellent negative predictive value, their positive predictive value is poor. Extracellular vesicles released by the placenta into the maternal circulation, syncytiotrophoblast membrane extracellular vesicles (STB-EVs), have been identified as being involved in PE with the potential to act as liquid biopsies. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
maternal placenta perfusate
Sample origin
Preeclampsia
Focus vesicles
small extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD63/ PLAP/ CD9
non-EV: Cytochrome C
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
maternal placenta perfusate
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Sorvall TST28.39
Pelleting: speed (g)
150,000
Wash: volume per pellet (ml)
45
Wash: time (min)
120
Wash: Rotor Type
Sorvall TST28.39
Wash: speed (g)
150,000
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
5780-10648
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD63/ PLAP
Not detected EV-associated proteins
CD9
Not detected contaminants
Cytochrome C
Flow cytometry
Type of Flow cytometry
A BD LSRII flow cytometer (BD Biosciences) with a blue, violet, and red laser
Calibration bead size
0.11/ 0.18/ 0.24/ 0.30/ 0.50/ 0.59/ 0.88/ 1.30
Antibody details provided?
No
Proteomics database
PRIDE
Characterization: RNA analysis
RNA analysis
Type
(RT)-(q)PCR/ RNA-sequencing/ Capillary electrophoresis (e.g. Bioanalyzer)
Database
NCBI geo
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
479
EV concentration
Yes
Particle yield
2.2E11-7.2E11
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210302 1/2 Homo sapiens MKN74 (d)(U)C
Filtration
qEV
Poças J 2023 78%

Study summary

Full title
All authors
Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A
Journal
Proc Natl Acad Sci U S A
Abstract
Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
qEV
Protein markers
EV: Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4
non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MKN74
EV-harvesting Medium
Serum free medium
Cell viability (%)
88
Cell count
73000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
160
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
160
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Filtration steps
0.2 or 0.22 µm
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD63/ CD81/ HSP70/ SDCBP/ SDC4
Not detected contaminants
CytochromeC
Proteomics database
PRIDE Proteomics
Detected contaminants
Calreticulin/ GM130/ PMP70/ Prohibitin
Not detected contaminants
Albumin/ Argonaute2/ CytochromeC/ Tubulin1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
102.1
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.90E+08
EM
EM-type
Transmission-EM/ Immuno-EM
EM protein
SDC4
Image type
Close-up, Wide-field
EV210302 2/2 Homo sapiens MKN74 (d)(U)C
Filtration
qEV
Poças J 2023 78%

Study summary

Full title
All authors
Poças J, Marques C, Gomes C, Otake AH, Pinto F, Ferreira M, Silva T, Faria-Ramos I, Matos R, Ribeiro AR, Senra E, Cavadas B, Batista S, Maia J, Macedo JA, Lima L, Afonso LP, Ferreira JA, Santos LL, Polónia A, Osório H, Belting M, Reis CA, Costa-Silva B, Magalhães A
Journal
Proc Natl Acad Sci U S A
Abstract
Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options (show more...)Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
SDC4 KO
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
qEV
Protein markers
EV: Alix/ CD9/ CD81/ HSP70/ SDCBP
non-EV: CytochromeC/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Albumin/ Argonaute2/ Tubulin1
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MKN74
EV-harvesting Medium
Serum free medium
Cell viability (%)
92
Cell count
74000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
160
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
160
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Filtration steps
0.2 or 0.22 µm
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
per million cells
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD9/ CD81/ HSP70/ SDCBP
Not detected contaminants
CytochromeC/ Tubulin1
Proteomics database
PRIDE Proteomics
Detected contaminants
Calreticulin/ GM130/ PMP70/ Prohibitin
Not detected contaminants
Albumin/ Argonaute2/ CytochromeC/ Tubulin1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
86
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 6.75E+08
EM
EM-type
Transmission-EM/ Immuno-EM
EM protein
SDC4
Image type
Close-up, Wide-field
EV230981 6/6 Mus musculus Blood plasma (d)(U)C
DG
qEVoriginal/70nm
André-Grégoire G 2023 75%

Study summary

Full title
All authors
André-Grégoire G, Roux Q, Gavard J
Journal
STAR Protoc
Abstract
Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological co (show more...)Circulating extracellular vesicles (EVs) could serve for the surveillance of diverse pathological conditions. We present a protocol for enriching and isolating plasma EVs from mouse blood. We describe steps for employing ultracentrifugation, size-exclusion chromatography, and density gradients, required for further quantitative and qualitative analysis. We detail the procedure for retrieving optimal volume of blood while preserving its integrity and avoiding hemolysis. We also describe the preparation of EVs from this complex fluid containing soluble proteins, aggregates, and lipoprotein particles. For complete details on the use and execution of this protocol, please refer to André-Grégoire et al. (2022).. (hide)
EV-METRIC
75% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported