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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
Experiment number
  • Experiments differ in Isolation/particle analysis
Experiment number
  • Experiments differ in Isolation/particle analysis
Experiment number
  • Experiments differ in Sample type
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in sample origin
Experiment number
  • Experiments differ in Isolation/particle analysis
Experiment number
  • Experiments differ in Isolation/particle analysis
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  • Experiments differ in Isolation/particle analysis
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  • Experiments differ in Sample type
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Experiment number
  • Experiments differ in Sample type
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV200179 3/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
DG
UF
Ouyang, Yingshi 2016 88%

Study summary

Full title
All authors
Yingshi Ouyang, Avraham Bayer, Tianjiao Chu, Vladimir A Tyurin, Valerian E Kagan, Adrian E Morelli, Carolyn B Coyne, Yoel Sadovsky
Journal
Placenta
Abstract
Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among (show more...)Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
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
Density gradient
Ultrafiltration
Protein markers
EV: TSG101/ CD63/ Syntenin-1
non-EV: Argonaute2
Proteomics
yes
EV density (g/ml)
1.08-1.10
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Primary human trophoblast
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Overnight, 100000g or commercial
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
6
Highest density fraction
40
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
Not specified
Speed (g)
100000
Duration (min)
Overnight
Fraction volume (mL)
.
Fraction processing
Ultrafiltration
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100 kda
Membrane type
PES
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
CD63/ Syntenin-1/ TSG101
Not detected contaminants
Argonaute2
Proteomics database
Yes: Data and Specimen Hub
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mode
Reported size (nm)
80-90nm
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV160001 1/1 Mus musculus Cell culture supernatant DG
(d)(U)C
Stremersch S 2016 87%

Study summary

Full title
All authors
Stremersch S, Vandenbroucke RE, Van Wonterghem E, Hendrix A, De Smedt SC, Raemdonck K
Journal
J Control Release
Abstract
Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natu (show more...)Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natural carriers for biomolecule transfer between cells. This unique feature rationalizes their exploitation as bio-inspired drug delivery systems. However, the therapeutic application of ELVs is hampered by the lack of efficient and reproducible drug loading methods, in particular for therapeutic macromolecules. To overcome this limitation, we present a generic method to attach siRNA to the surface of isolated ELVs by means of a cholesterol anchor. Despite a feasible uptake in both a dendritic and lung epithelial cell line, B16F10- and JAWSII-derived ELVs were unable to functionally deliver the associated small RNAs, neither exogenous cholesterol-conjugated siRNA nor endogenous miRNA derived from the melanoma producer cell. The latter results were confirmed both for purified ELVs and ELVs delivered via a transwell co-culture set-up. In contrast, simple anionic fusogenic liposomes were able to induce a marked siRNA-mediated gene knockdown under equal experimental conditions, both indicating successful cytosolic delivery of surface-bound cholesterol-conjugated siRNA and further underscoring the incapacity of the here evaluated ELVs to guide cytosolic delivery of small RNAs. In conclusion, we demonstrate that a more in-depth understanding of the biomolecular delivery mechanism and specificity is required before ELVs can be envisioned as a generic siRNA carrier. (hide)
EV-METRIC
87% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome-like vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: CD81/ HSP70/ beta-actin/ CD63
non-EV: GM130/ Calreticulin
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
B16F10, JAWSII
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
Ultrafiltration (MWCO 300 kDa)
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Density medium
115.5
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
0.125
Highest density fraction
0.5
Sample volume (mL)
1
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 55 Ti
Speed (g)
200000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
150
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
115.5
Pelleting-wash: volume per pellet (mL)
5
Pelleting-wash: duration (min)
70
Pelleting-wash: rotor type
115.5
Pelleting-wash: speed (g)
SW 55 Ti
Pelleting-wash: adjusted k-factor
115.5
EV-subtype
Used subtypes
NO
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, HSP70, beta-actin
Flow cytometry specific beads
Antibody details provided?
No
Selected surface protein(s)
CD63
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-300
EV concentration
Yes
Particle yield
1000
EM
EM-type
Cryo-EM
Image type
Close-up
EV220152 3/6 Mus musculus Cell culture supernatant (d)(U)C
DG
Vardaki I 2016 78%

Study summary

Full title
All authors
Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T
Journal
Oncotarget
Abstract
Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (hide)
EV-METRIC
78% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
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
Density gradient
Protein markers
EV: CD63/ TSG101/ Rab5/ integrin alpha2/ integrin beta1/ periostin/ N-cadherin/ LOXL3/ LOXL4/ Glypican-1/ Glypican-4/ V-ATPase/ Alix/ beta-catenin/ E-cadherin/ Syndecan-4/ Vimentin/ GADPH/ AIF
non-EV: None
Proteomics
yes
EV density (g/ml)
1.12-1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
67NR
EV-harvesting Medium
EV-depleted medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: speed (g)
120000
Wash: time (min)
120
Wash: speed (g)
120000
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.2 M
Highest density fraction
2 M
Orientation
Top-down
Speed (g)
120000
Duration (min)
1200
Fraction processing
Centrifugation
Pelleting: duration (min)
120
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ TSG101/ Rab5/ integrin alpha2/ integrin beta1/ periostin/ N-cadherin/ LOXL3/ LOXL4/ Glypican-1/ Glypican-4/ V-ATPase/ Alix
Not detected EV-associated proteins
beta-catenin/ E-cadherin/ Syndecan-4/ Vimentin/ GADPH/ AIF
Flow cytometry aspecific beads
Antibody details provided?
Yes
Flow cytometry specific beads
Antibody details provided?
No
Detected EV-associated proteins
CD63
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
89
EV concentration
Yes
EM
EM-type
Transmission­-EM
Image type
Close-up, Wide-field
EV220152 4/6 Mus musculus Cell culture supernatant (d)(U)C
DG
Vardaki I 2016 78%

Study summary

Full title
All authors
Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T
Journal
Oncotarget
Abstract
Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (hide)
EV-METRIC
78% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
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
Density gradient
Protein markers
EV: CD63/ TSG101/ Rab5/ Alix/ beta-catenin/ integrin alpha2/ integrin beta1/ periostin/ E-cadherin/ LOXL4/ Glypican-1/ V-ATPase/ Alix/ Vimentin/ GAPDH/ AIF/ N-Cadherin/ LOXL3/ Syndecan-4/ Glypican-4/ LOXL3/ N-Cadherin
non-EV: None
Proteomics
yes
EV density (g/ml)
1.12-1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
4T1
EV-harvesting Medium
EV-depleted medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: speed (g)
120000
Wash: time (min)
120
Wash: speed (g)
120000
Density gradient
Only used for validation of main results
Yes
Lowest density fraction
0.2 M
Highest density fraction
2 M
Orientation
Top-down
Speed (g)
120000
Duration (min)
1200
Fraction processing
Centrifugation
Pelleting: duration (min)
120
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ TSG101/ Rab5/ Alix/ beta-catenin/ integrin alpha2/ integrin beta1/ periostin/ E-cadherin/ LOXL4/ Glypican-1/ V-ATPase/ Alix
Not detected EV-associated proteins
Vimentin/ GAPDH/ AIF/ N-Cadherin/ LOXL3/ Syndecan-4/ Glypican-4/ LOXL3/ N-Cadherin
Flow cytometry aspecific beads
Antibody details provided?
Yes
Flow cytometry specific beads
Antibody details provided?
No
Detected EV-associated proteins
CD81
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
102
EV concentration
Yes
EM
EM-type
Transmission­-EM
Image type
Close-up, Wide-field
EV210135 1/2 Homo sapiens Cell culture supernatant DG
(d)(U)C
UF
Stremersch, Stephan 2016 78%

Study summary

Full title
All authors
Stephan Stremersch, Monica Marro, Bat-El Pinchasik, Pieter Baatsen, An Hendrix, Stefaan C De Smedt, Pablo Loza-Alvarez, Andre G Skirtach, Koen Raemdonck, Kevin Braeckmans
Journal
Small
Abstract
Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention f (show more...)Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis. (hide)
EV-METRIC
78% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
UF
Protein markers
EV: CD81/ HSP70/ CD63/ actin
non-EV: None
Proteomics
no
EV density (g/ml)
1.14
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-­related methods/New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Red blood cells
EV-harvesting Medium
Serum free medium
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
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
12.5%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
Not specified
Sample volume (mL)
Not spec
Orientation
Top-down
Rotor type
SW 55 Ti
Speed (g)
150000
Duration (min)
900
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
150
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
150000
Ultra filtration
Cut-off size (kDa)
30
Membrane type
Not specified
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ actin/ HSP70/ CD81
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
170
Particle yield
Not reported
EM
EM-type
Cryo-­EM
Image type
Wide-field
EV concentration
Not
EV210135 2/2 Mus musculus Cell culture supernatant DG
(d)(U)C
UF
Stremersch, Stephan 2016 78%

Study summary

Full title
All authors
Stephan Stremersch, Monica Marro, Bat-El Pinchasik, Pieter Baatsen, An Hendrix, Stefaan C De Smedt, Pablo Loza-Alvarez, Andre G Skirtach, Koen Raemdonck, Kevin Braeckmans
Journal
Small
Abstract
Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention f (show more...)Exosome-like vesicles (ELVs) are a novel class of biomarkers that are receiving a lot of attention for the detection of cancer at an early stage. In this study the feasibility of using a surface enhanced Raman spectroscopy (SERS) based method to distinguish between ELVs derived from different cellular origins is evaluated. A gold nanoparticle based shell is deposited on the surface of ELVs derived from cancerous and healthy cells, which enhances the Raman signal while maintaining a colloidal suspension of individual vesicles. This nanocoating allows the recording of SERS spectra from single vesicles. By using partial least squares discriminant analysis on the obtained spectra, vesicles from different origin can be distinguished, even when present in the same mixture. This proof-of-concept study paves the way for noninvasive (cancer) diagnostic tools based on exosomal SERS fingerprinting in combination with multivariate statistical analysis. (hide)
EV-METRIC
78% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
UF
Protein markers
EV: CD81/ HSP70/ CD63/ actin
non-EV: None
Proteomics
no
EV density (g/ml)
1.14
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-­related methods/New methodological development
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
B16F10
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Other preparation;Ultrafiltration
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
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
12.5%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
Not specified
Sample volume (mL)
Not spec
Orientation
Top-down
Rotor type
SW 55 Ti
Speed (g)
150000
Duration (min)
900
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
150
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
150000
Ultra filtration
Cut-off size (kDa)
30
Membrane type
Not specified
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ actin/ HSP70/ CD81
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
120
Particle yield
Not reported
EM
EM-type
Cryo-­EM
Image type
Wide-field
EV concentration
Not
EV210035 2/2 Homo sapiens Cell culture supernatant (d)(U)C
DG
Filtration
van Dommelen, Susan M 2016 78%

Study summary

Full title
All authors
Susan M van Dommelen, Roy van der Meel, Wouter W van Solinge, Maria Coimbra, Pieter Vader, Raymond M Schiffelers
Journal
Nanomedicine
Abstract
Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their co (show more...)Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). Materials & methods: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. Results: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. Conclusion: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior. Keywords: EGFR; biomarker; cancer therapy; cetuximab; diagnostics; exosomes; extracellular vesicles. (hide)
EV-METRIC
78% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Cetuximab
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
Filtration
Protein markers
EV: TSG101/ Alix/ CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ Cetuximab
non-EV: Lamin-A/ Lamin-C/ ATP5-A/ Tom20
Proteomics
no
EV density (g/ml)
1.10-1.21
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
A-431
EV-harvesting Medium
Serum free medium
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)
70
Pelleting: rotor type
JA-30.50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
JA-30.50
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
0.4
Highest density fraction
2.5
Total gradient volume, incl. sample (mL)
Not specified
Sample volume (mL)
Not spec
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ Cetuximab/ TSG101/ Alix
Not detected contaminants
Lamin-A/ Lamin-C/ ATP5-A/ Tom20
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV200179 4/4 Homo sapiens Blood plasma (d)(U)C
Filtration
DG
UF
PEG precipitaton
Gelatin-sepharose chromatograhy
Ouyang, Yingshi 2016 75%

Study summary

Full title
All authors
Yingshi Ouyang, Avraham Bayer, Tianjiao Chu, Vladimir A Tyurin, Valerian E Kagan, Adrian E Morelli, Carolyn B Coyne, Yoel Sadovsky
Journal
Placenta
Abstract
Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among (show more...)Introduction: Primary human trophoblasts release a repertoire of extracellular vesicles (EVs). Among them are nano-sized exosomes, which we found to suppress the replication of a wide range of diverse viruses. These exosomes contain trophoblastic microRNAs (miRNAs) that are expressed from the chromosome 19 miRNA cluster and exhibit antiviral properties. Here, we report our investigation of the cargo of placental EVs, focusing on the composition and the antiviral properties of exosomes, microvesicles, and apoptotic blebs. Methods: We isolated EVs using ultracentrifugation and defined their purity using immunoblotting, electron microscopy, and nanoparticle tracking. We used liquid chromatography-electrospray ionization-mass spectrometry, protein mass spectrometry, and miRNA TaqMan card PCR to examine the phospholipids, proteins, and miRNA cargo of trophoblastic EVs and an in vitro viral infection assay to assess the antiviral properties of EVs. Results: We found that all three EV types contain a comparable repertoire of miRNA. Interestingly, trophoblastic exosomes harbor a protein and phospholipid profile that is distinct from that of microvesicles or apoptotic blebs. Functionally, trophoblastic exosomes exhibit the highest antiviral activity among the EVs. Consistently, plasma exosomes derived from pregnant women recapitulate the antiviral effect of trophoblastic exosomes derived from in vitro cultures of primary human trophoblasts. Discussion: When compared to other trophoblastic EVs, exosomes exhibit a unique repertoire of proteins and phospholipids, but not miRNAs, and a potent viral activity. Our work suggests that human trophoblastic EVs may play a key role in maternal-placental-fetal communication. (hide)
EV-METRIC
75% (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
Blood plasma
Sample origin
Healthy pregnant
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
Density gradient
Ultrafiltration
PEG precipitaton
Gelatin-sepharose chromatograhy
Protein markers
EV: CD63
non-EV: Fibronectin
Proteomics
yes
EV density (g/ml)
1.08-1.10
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Continuous
Lowest density fraction
6
Highest density fraction
30
Total gradient volume, incl. sample (mL)
14
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
Not specified
Speed (g)
10000
Duration (min)
Overnight
Fraction volume (mL)
.
Fraction processing
Ultrafiltration
Pelleting: volume per fraction
.
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100 kda
Membrane type
PES
Other
Name other separation method
PEG precipitaton
Other
Name other separation method
Gelatin-sepharose chromatograhy
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
CD63
Not detected contaminants
Fibronectin
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mode
Reported size (nm)
80-90nm
EV concentration
Yes
EV210487 1/2 Mus musculus Cell culture supernatant DG
(d)(U)C
UF
Filtration
Bosch S 2016 67%

Study summary

Full title
All authors
Bosch S, de Beaurepaire L, Allard M, Mosser M, Heichette C, Chrétien D, Jegou D, Bach JM
Journal
Sci Rep
Abstract
Exosomes are important mediators in intercellular communication. Released by many cell types, they t (show more...)Exosomes are important mediators in intercellular communication. Released by many cell types, they transport proteins, lipids, and nucleic acids to distant recipient cells and contribute to important physiopathological processes. Standard current exosome isolation methods based on differential centrifugation protocols tend to induce aggregation of particles in highly concentrated suspensions and freezing of exosomes can induce damage and inconsistent biological activity. Trehalose is a natural, non-toxic sugar widely used as a protein stabilizer and cryoprotectant by the food and drug industry. Here we report that addition of 25 mM trehalose to pancreatic beta-cell exosome-like vesicle isolation and storage buffer narrows the particle size distribution and increases the number of individual particles per microgram of protein. Repeated freeze-thaw cycles induce an increase in particle concentration and in the width of the size distribution for exosome-like vesicles stored in PBS, but not in PBS 25 mM trehalose. No signs of lysis or incomplete vesicles were observed by cryo-electron tomography in PBS and trehalose samples. In macrophage immune assays, beta-cell extracellular vesicles in trehalose show consistently higher TNF-alpha cytokine secretion stimulation indexes suggesting improved preservation of biological activity. The addition of trehalose might be an attractive means to standardize experiments in the field of exosome research and downstream applications. (hide)
EV-METRIC
67% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Ultrafiltration
Filtration
Protein markers
EV: CD81/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
MIN6
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.1
Sample volume (mL)
0.60
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
180
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
No
Antibody dilution provided?
Yes
Detected EV-associated proteins
CD63/ CD81
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNA analysis
Type
Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
113
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210487 2/2 Mus musculus Cell culture supernatant DG
(d)(U)C
UF
Filtration
Bosch S 2016 67%

Study summary

Full title
All authors
Bosch S, de Beaurepaire L, Allard M, Mosser M, Heichette C, Chrétien D, Jegou D, Bach JM
Journal
Sci Rep
Abstract
Exosomes are important mediators in intercellular communication. Released by many cell types, they t (show more...)Exosomes are important mediators in intercellular communication. Released by many cell types, they transport proteins, lipids, and nucleic acids to distant recipient cells and contribute to important physiopathological processes. Standard current exosome isolation methods based on differential centrifugation protocols tend to induce aggregation of particles in highly concentrated suspensions and freezing of exosomes can induce damage and inconsistent biological activity. Trehalose is a natural, non-toxic sugar widely used as a protein stabilizer and cryoprotectant by the food and drug industry. Here we report that addition of 25 mM trehalose to pancreatic beta-cell exosome-like vesicle isolation and storage buffer narrows the particle size distribution and increases the number of individual particles per microgram of protein. Repeated freeze-thaw cycles induce an increase in particle concentration and in the width of the size distribution for exosome-like vesicles stored in PBS, but not in PBS 25 mM trehalose. No signs of lysis or incomplete vesicles were observed by cryo-electron tomography in PBS and trehalose samples. In macrophage immune assays, beta-cell extracellular vesicles in trehalose show consistently higher TNF-alpha cytokine secretion stimulation indexes suggesting improved preservation of biological activity. The addition of trehalose might be an attractive means to standardize experiments in the field of exosome research and downstream applications. (hide)
EV-METRIC
67% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Trehalose
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
(Differential) (ultra)centrifugation
Ultrafiltration
Filtration
Protein markers
EV: CD81/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
MIN6
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.1
Sample volume (mL)
0.60
Orientation
Top-down
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
180
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
No
Antibody dilution provided?
Yes
Detected EV-associated proteins
CD63/ CD81
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNA analysis
Type
Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
111
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV210101 4/6 Homo sapiens Urine (d)(U)C
Filtration
SEC (non-commercial)
Welton, Joanne Louise 2016 67%

Study summary

Full title
All authors
Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton
Journal
J Extracell Vesicles
Abstract
Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: Alix/ TSG101/ LAMP2A
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
2.8
Sample volume/column (mL)
0.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Other;Spectrophotometry
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ LAMP2A/ TSG101
Not detected contaminants
Albumin
Detected EV-associated proteins
SOMAscan multiplex assay
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
117.75
EV concentration
Yes
Particle yield
As the number of particles per µg protein;Yes, other: 20000000000
EM
EM-type
Cryo-EM
Image type
Close-up
EV200134 2/2 Homo sapiens Blood plasma DG
(d)(U)C
Filtration
Salomon, Carlos 2016 67%

Study summary

Full title
All authors
Carlos Salomon, Katherin Scholz-Romero, Suchismita Sarker, Emma Sweeney, Miharu Kobayashi, Paula Correa, Sherri Longo, Gregory Duncombe, Murray D Mitchell, Gregory E Rice, Sebastian E Illanes
Journal
Diabetes
Abstract
Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) d (show more...)Although there is significant interest in elucidating the role of placenta-derived exosomes (PdEs) during pregnancy, the exosomal profile in pregnancies complicated by gestational diabetes mellitus (GDM) remains to be established. The aim of this study was to compare the gestational-age profile of PdEs in maternal plasma of GDM with normal pregnancies and to determine the effect of exosomes on cytokine release from human umbilical vein endothelial cells. A prospective cohort of patients was sampled at three time points during pregnancy for each patient (i.e., 11-14, 22-24, and 32-36 weeks' gestation). A retrospective stratified study design was used to quantify exosomes present in maternal plasma of normal (n = 13) and GDM (n = 7) pregnancies. Gestational age and pregnancy status were identified as significant factors contributing to variation in plasma exosome concentration (ANOVA, P < 0.05). Post hoc analyses established that PdE concentration increased during gestation in both normal and GDM pregnancies; however, the increase was significantly greater in GDM (∼2.2-fold, ∼1.5-fold, and ∼1.8-fold greater at each gestational age compared with normal pregnancies). Exosomes isolated from GDM pregnancies significantly increased the release of proinflammatory cytokines from endothelial cells. Although the role of exosomes during GDM remains to be fully elucidated, exosome profiles may be of diagnostic utility for screening asymptomatic populations. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Gestational diabetes mellitus
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
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: PLAP/ CD63/ TSG101
non-EV: None
Proteomics
no
EV density (g/ml)
1.122-1.156
Show all info
Study aim
Function
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: time(min)
120
Pelleting: rotor type
T-8100
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
120
Wash: Rotor Type
T-8100
Wash: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
14.5mL
Sample volume (mL)
0.5mL
Orientation
Top-down
Rotor type
T-8100
Speed (g)
100000
Duration (min)
1200
Fraction processing
Centrifugation
Pelleting: duration (min)
120
Pelleting: rotor type
T-8100
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ TSG101
ELISA
Antibody details provided?
No
Detected EV-associated proteins
PLAP
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
100 - 108nm
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV160004 2/5 Equus caballus Synovial fluid DG
(d)(U)C
Boere J 2016 66%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
66% (63rd 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
Synovial fluid
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
71.08 (pelleting)
Protein markers
EV: Annexin-A1/ CD90/Thy1.1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
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: time(min)
120
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
71.08
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0M
Highest density fraction
1.4M
Sample volume (mL)
1.5
Orientation
Bottom-up (sample migrates upwards)
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
60
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
138.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Annexin-A1, CD90/Thy1.1
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
20-200
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.
EV160004 5/5 Equus caballus Synovial fluid DG
(d)(U)C
Boere J 2016 66%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
66% (63rd 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
Synovial fluid
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
1421 (pelleting)
Protein markers
EV: Annexin-A1/ CD90/Thy1.1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
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: time(min)
120
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
10000
Pelleting: adjusted k-factor
1421.
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0M
Highest density fraction
1.4M
Sample volume (mL)
1.5
Orientation
Bottom-up (sample migrates upwards)
Rotor type
MLS-50
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
60
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
138.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Annexin-A1, CD90/Thy1.1
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
Report size (nm)
20-200
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.
EV200044 1/1 Homo sapiens Blood plasma Precipitation
DG
Zhang YN 2016 63%

Study summary

Full title
All authors
Zhang YN, Vernooij F, Ibrahim I, Ooi S, Gijsberts CM, Schoneveld AH, Sen KW, den Ruijter HM, Timmers L, Richards AM, Jong CT, Mazlan I, Wang JW, Lam CS, de Kleijn DP
Journal
PLoS One
Abstract
BACKGROUND: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma (show more...)BACKGROUND: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma extracellular vesicle (EV) -levels of these proteins have been reported to be associated with systemic vascular events. Evidence is accumulating that inflammatory processes may play a pivotal role both in systemic vascular events and in heart failure. Therefore, we studied the association between plasma extracellular vesicle SerpinF2-, SerpinG1-, CystatinC and CD14-levels and the occurrence of acute heart failure in patients. METHODS AND RESULT: Extracellular vesicle protein levels of SerpinG1, SerpinF2, CystatinC and CD14 were measured in an observational study of 404 subjects presenting with dysponea at the emergency department (4B-cohort). Plasma extracellular vesicles were precipitated in a total extracellular vesicles (TEX)-fraction and in separate LDL- and HDL-subfractions. Extracellular vesicle protein levels were measured with a quantitative immune assay in all 3 precipitates. Out of 404 subjects, 141 (35%) were diagnosed with acutely decompensated heart failure. After correction for confounders (including comorbidities and medications), levels of CD14 in the HDL-fraction (OR 1.53, p = 0.01), SerpinF2 in the TEX-and LDL-fraction (ORs respectively 0.71 and 0.65, p<0.05) and SerpinG1 in the TEX-fraction (OR 1.55, p = 0.004) were statistically significantly related to heart failure. Furthermore, extracellular vesicle CD14- and SerpinF2-levels were significantly higher in heart failure patients with preserved ejection fraction than in those with reduced ejection fraction. CONCLUSION: Extracellular vesicle levels of CD14, SerpinG1 and SerpinF2 are associated with the occurrence of heart failure in subjects suspected for acute heart failure, suggesting common underlying pathophysiological mechanisms for heart failure and vascular events. (hide)
EV-METRIC
63% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
Healthy test subject
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
Precipitation
Density gradient
Protein markers
EV: CD14/ SerpinC1/ SerpinG1/ CystC/ SerpinF2
non-EV: None
Proteomics
no
EV density (g/ml)
1.02-1.17
Show all info
Study aim
Biomarker/New methodological development
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10.5
Sample volume (mL)
0.5
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
200000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
8
Pelleting: duration (min)
60
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
200000
Other
Name other separation method
Precipitation
Other
Name other separation method
Density gradient
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD14/ SerpinC1/ SerpinG1/ CystC/ SerpinF2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-120
EV210035 1/2 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
van Dommelen, Susan M 2016 56%

Study summary

Full title
All authors
Susan M van Dommelen, Roy van der Meel, Wouter W van Solinge, Maria Coimbra, Pieter Vader, Raymond M Schiffelers
Journal
Nanomedicine
Abstract
Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their co (show more...)Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). Materials & methods: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. Results: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. Conclusion: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior. Keywords: EGFR; biomarker; cancer therapy; cetuximab; diagnostics; exosomes; extracellular vesicles. (hide)
EV-METRIC
56% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ Alix/ Cetuximab/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ CD9
non-EV: Lamin-A/ Lamin-C/ ATP5-A/ Tom20
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
A-431
EV-harvesting Medium
Serum free medium
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)
70
Pelleting: rotor type
JA-30.50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
JA-30.50
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ TSG101/ Alix
Not detected EV-associated proteins
Cetuximab
Not detected contaminants
Lamin-A/ Lamin-C/ ATP5-A/ Tom20
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV160008 1/3 Rattus norvegicus Cell culture supernatant (d)(U)C Cianciaruso C 2016 55%

Study summary

Full title
All authors
Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S
Journal
Diabetes
Abstract
The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide)
EV-METRIC
55% (83rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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
(d)(U)C
Protein markers
EV: GAD67/ GAD65/ Insulin/ PDI/ Flotillin-1/ CD9/ IA-2
non-EV: Gp96/ Calreticulin
Proteomics
yes
Show all info
Study aim
Function, Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
EV-producing cells
Primary pancreatic islets
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
NA
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: time(min)
70
Pelleting: speed (g)
110000
Wash: time (min)
70
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1, GAD65, GAD67, IA-2, PDI
Not detected contaminants
Calreticulin, Gp96
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mode
Reported size (nm)
139
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
120
EV160005 1/2 Homo sapiens Bronchoalveolar lavage fluid (d)(U)C
Filtration
Héliot A 2016 55%

Study summary

Full title
All authors
Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ
Journal
Int J Hyg Environ Health
Abstract
Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide)
EV-METRIC
55% (25th 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
Bronchoalveolar lavage fluid
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Adj. k-factor
126 (pelleting) / 126 (washing)
Protein markers
EV: CD81/ CD63/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker
Sample
Species
Homo sapiens
Sample Type
Bronchoalveolar lavage fluid
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
126.0
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
110000
Wash: adjusted k-factor
126.0
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, CD81
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution;Mean
Reported size (nm)
138.1±2.2
EV concentration
Yes
Particle yield
2.48E+11 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-200
Extra information
UC rotors added to the EV-TRACK entry after publication
EV160005 2/2 Homo sapiens Bronchoalveolar lavage fluid (d)(U)C
Filtration
Héliot A 2016 55%

Study summary

Full title
All authors
Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ
Journal
Int J Hyg Environ Health
Abstract
Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide)
EV-METRIC
55% (25th 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
Bronchoalveolar lavage fluid
Sample origin
Smokers
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
(d)(U)C
Filtration
Adj. k-factor
126 (pelleting) / 126 (washing)
Protein markers
EV: CD81/ CD63/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker
Sample
Species
Homo sapiens
Sample Type
Bronchoalveolar lavage fluid
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
126.0
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
110000
Wash: adjusted k-factor
126.0
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, CD81
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution;Mean
Reported size (nm)
139.8±3.3
EV concentration
Yes
Particle yield
1.94E+11 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-200
Extra information
UC rotors added to the EV-TRACK entry after publication
EV160004 1/5 Equus caballus Synovial fluid DG
(d)(U)C
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (40th 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
Synovial fluid
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
83.68 (pelleting)
Protein markers
EV: CD44/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
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
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
200000
Pelleting: adjusted k-factor
83.68
Density gradient
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Antibody details provided?
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
EV160004 3/5 Equus caballus Synovial fluid DG
(d)(U)C
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (40th 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
Synovial fluid
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
167.3 (pelleting)
Protein markers
EV: CD44/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
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)
65
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
167.3
Density gradient
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Antibody details provided?
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
EV160004 4/5 Equus caballus Synovial fluid DG
(d)(U)C
Boere J 2016 55%

Study summary

Full title
All authors
Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide)
EV-METRIC
55% (40th 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
Synovial fluid
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
1673 (pelleting)
Protein markers
EV: CD44/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker, New methodological development
Sample
Species
Equus caballus
Sample Type
Synovial fluid
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: time(min)
35
Pelleting: rotor type
SW 60 Ti
Pelleting: speed (g)
10000
Pelleting: adjusted k-factor
1673.
Density gradient
Type
Continuous
Number of initial discontinuous layers
15
Highest density fraction
0.51
Sample volume (mL)
1.75
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
19
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD44
Flow cytometry
Type of Flow cytometry
BD-Influx
Hardware adjustments
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1,0.2
Antibody details provided?
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD-Influx
Hardware adjustment
optimized jet-in-air-based BD Influx flow cytometer
Calibration bead size
0.1;0.2
EV concentration
Yes
Particle yield
7.00E+08 particles/ml start sample
Extra information
Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).
EV210488 1/11 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
DG
van Eijndhoven MA 2016 50%

Study summary

Full title
All authors
van Eijndhoven MA, Zijlstra JM, Groenewegen NJ, Drees EE, van Niele S, Baglio SR, Koppers-Lalic D, van der Voorn H, Libregts SF, Wauben MH, de Menezes RX, van Weering JR, Nieuwland R, Visser L, van den Berg A, de Jong D, Pegtel DM
Journal
JCI insight
Abstract
Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive bioma (show more...)Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID). (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Density gradient
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
15
Lowest density fraction
0.4M
Highest density fraction
2M
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
840-1200
Fraction volume (mL)
1
Fraction processing
None
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
1.5
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ RNA sequencing/ Capillary electrophoresis (e.g. Bioanalyzer)
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
50-500
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Influx (Becton Dickinson)
Hardware adjustment
BD Influx cell sorter Influx cell sorter , which was equipped by Cytopeia and BD with: 488nm laser (200 mW)/ 561-nm laser (100 mW)/ 635-nm CUBE laser (30 mW) / SPD, containing a Mitutoyo Plan Apo 20 lens (NA 0.42), a 0.7-mm pinhole and a polarization unit with two FSC PMTs/ Optical filters/ amplifier board with 2 'fast' preamplifiers (0.12 s) and 6 'slow' preamplifiers (1.2 s)/ and Spigot software, version 6.1.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV210101 3/6 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
Filtration
Welton, Joanne Louise 2016 50%

Study summary

Full title
All authors
Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton
Journal
J Extracell Vesicles
Abstract
Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
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
(d)(U)C
Size-exclusion chromatography (non-commercial)
Filtration
Protein markers
EV: CD81/ CD9
non-EV: Albumin/ ApoB
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-110
Pelleting: speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
12
Sample volume/column (mL)
1.5
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Other;Spectrophotometry
ELISA
Antibody details provided?
No
Detected EV-associated proteins
CD81/ CD9
Detected contaminants
ApoB
Detected EV-associated proteins
SOMAscan multiplex assay
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
86.47
EV concentration
Yes
Particle yield
As the number of particles per µg protein;Yes, other: 5000000000
EM
EM-type
Cryo-EM
Image type
Close-up
EV160007 1/3 Homo sapiens Blood plasma (d)(U)C
Filtration
SEC
Hong CS 2016 50%

Study summary

Full title
All authors
Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL
Journal
J Extracell Vesicles
Abstract
OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
Acute myeloid leukemia
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
(d)(U)C
Filtration
SEC
Protein markers
EV: TSG101/ PD-1/ CD123/ CD96/ CD44/ CD34/ Pro-TGFbeta1 LAP/ Pro-TGFbeta1LAP/ PD-L1/ CLL-1/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
66
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, TSG101, CD44, CD34, CD123, CD96, CLL-1, Pro-TGFbeta1 LAP, PD-1, PD-L1
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
77-92
EV concentration
Yes
Particle yield
8.90E+10 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160007 2/3 Homo sapiens Blood plasma (d)(U)C
Filtration
SEC
Hong CS 2016 50%

Study summary

Full title
All authors
Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL
Journal
J Extracell Vesicles
Abstract
OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
Head and neck squamous cell carcinoma
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
(d)(U)C
Filtration
SEC
Protein markers
EV: TSG101/ CD39/ PD-1/ CD73/ Cox2/ Fas/ FasL/ PD-L1/ HSP70/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
123
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, TSG101, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-1, PD-L1
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
73-76
EV concentration
Yes
Particle yield
1.10E+11 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160007 3/3 Homo sapiens Blood plasma (d)(U)C
Filtration
SEC
Hong CS 2016 50%

Study summary

Full title
All authors
Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL
Journal
J Extracell Vesicles
Abstract
OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
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
(d)(U)C
Filtration
SEC
Protein markers
EV: TSG101/ CD39/ CD123/ PD-1/ CD73/ Cox2/ Fas/ Pro-TGFbeta1 LAP/ Pro-TGFbeta1LAP/ FasL/ PD-L1/ CLL-1/ HSP70/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
32
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, TSG101, CD123, CLL-1, Pro-TGFbeta1 LAP, PD-1, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-L1
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
69-76
EV concentration
Yes
Particle yield
7.00E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
EV160000 1/1 Homo sapiens Milk DG
(d)(U)C
van Herwijnen MJ 2016 50%

Study summary

Full title
All authors
van Herwijnen MJ, Zonneveld MI, Goerdayal S, Nolte-'t Hoen EN, Garssen J, Stahl B, Maarten Altelaar AF, Redegeld FA, Wauben MH
Journal
Mol Cell Proteomics
Abstract
Breast milk contains several macromolecular components with distinctive functions, whereby milk fat (show more...)Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. (hide)
EV-METRIC
50% (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
Milk
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: Flotillin-1/ CD9
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Milk
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
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Sample volume (mL)
6.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900-1080
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
38.5
Pelleting: duration (min)
65
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
PMID previous EV protein analysis
25206958
Western Blot
Antibody details provided?
No
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1
Proteomics database
Yes
Characterization: Lipid analysis
No
PMID previous EV particle analysis
25206958
Extra information
A different gradient protocol was used in the publication of the same group that was referred to for additional protein and particle analysis of milk-derived extracellular vesicles (PMID: 25206958).
EV210034 1/14 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Au Yeung, Chi Lam 2016 45%

Study summary

Full title
All authors
Chi Lam Au Yeung, Ngai-Na Co, Tetsushi Tsuruga, Tsz-Lun Yeung, Suet-Ying Kwan, Cecilia S Leung, Yong Li, Edward S Lu, Kenny Kwan, Kwong-Kwok Wong, Rosemarie Schmandt, Karen H Lu, Samuel C Mok
Journal
Nat Commun
Abstract
Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the (show more...)Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer. (hide)
EV-METRIC
45% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. 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
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
Protein markers
EV: HSP70/ CD63
non-EV: GM130
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Primary cancer-associated adipocytes
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
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)
90
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
90
Wash: Rotor Type
Not specified
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
GM130
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;RNA sequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
70-130
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV220152 5/6 Homo sapiens Plasma (d)(U)C Vardaki I 2016 44%

Study summary

Full title
All authors
Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T
Journal
Oncotarget
Abstract
Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (hide)
EV-METRIC
44% (66th 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
Plasma
Sample origin
Breast cancer, localised
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
Protein markers
EV: E-Cadherin/ N-Cadherin/ Periostin/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
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
Yes
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
E-Cadherin/ N-Cadherin/ Periostin/ CD9
Flow cytometry aspecific beads
Antibody details provided?
Yes
Flow cytometry specific beads
Antibody details provided?
No
Detected EV-associated proteins
CD81
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
95
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 8.35e+9
EM
EM-type
Transmission­-EM
Image type
Close-up
EV220152 6/6 Homo sapiens Plasma (d)(U)C Vardaki I 2016 44%

Study summary

Full title
All authors
Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T
Journal
Oncotarget
Abstract
Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related (show more...)Breast cancer (BrCa) is the most frequent cancer type in women and a leading cause of cancer related deaths in the world. Despite the decrease in mortality due to better diagnostics and palliative care, there is a lack of prognostic markers of metastasis. Recently, the exploitation of liquid biopsies and in particular of the extracellular vesicles has shown promise in the identification of such prognostic markers. In this study we compared the proteomic content of exosomes derived from metastatic and non-metastatic human (MCF7 and MDA-MB-231) and mouse (67NR and 4T1) cell lines. We found significant differences not only in the amount of secreted exosomes but most importantly in the protein content of exosomes secreted from metastatic versus non-metastatic ones. We identified periostin as a protein that is enriched in exosomes secreted by metastatic cells and validated its presence in a pilot cohort of breast cancer patient samples with localized disease or lymph node (LN) metastasis. (hide)
EV-METRIC
44% (66th 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
Plasma
Sample origin
Breast cancer, lymph-node metastasis
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
Protein markers
EV: E-Cadherin/ N-Cadherin/ Periostin/ CD9
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
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
Yes
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
E-Cadherin/ N-Cadherin/ Periostin/ CD9
Flow cytometry aspecific beads
Antibody details provided?
Yes
Flow cytometry specific beads
Antibody details provided?
No
Detected EV-associated proteins
CD81
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
125
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 1.09e+10
EM
EM-type
Transmission­-EM
Image type
Close-up
EV220146 1/3 Homo sapiens Cell culture supernatant (d)(U)C
DG
Lee JE 2016 44%

Study summary

Full title
All authors
Lee JE, Moon PG, Cho YE, Kim YB, Kim IS, Park H, Baek MC
Journal
J Proteomics
Abstract
Cancer cell-derived extracellular vesicles have been linked to the pathogenesis of various cancers/ (show more...)Cancer cell-derived extracellular vesicles have been linked to the pathogenesis of various cancers/ however, the role of extracellular vesicles in tumorigenesis remains unclear. To identify extracellular vesicle proteins involved in cancer metastasis, quantitative proteomic analyses were performed on extracellular vesicles derived from two representative breast cancer cell lines: the less invasive MCF-7 and the invasive MDA-MB-231. Proteomic analysis allowed for the identification of 270 proteins in the extracellular vesicles. Here we report a new function of EDIL3 on extracellular vesicles, which are sufficient for enhancement of cell invasion and for acceleration of lung metastasis in vivo. This invasion is most likely mediated via the integrin-FAK signaling cascade in breast cancer cells. However, these effects are suppressed when EDIL3 is inactivated, providing evidence for a critical role of EDIL3 in development of cancer. Consistently, in human patients with metastatic breast cancer, the levels of EDIL3 on circulating extracellular vesicles are significantly elevated. This information is a remarkable breakthrough in understanding of the molecular mechanism underlying metastasis of breast cancer as well as in the research for cancer biomarkers using circulating extracellular vesicles. Furthermore, targeting EDIL3 on extracellular vesicles may lead to a new therapeutic option for treatment of breast cancer. (hide)
EV-METRIC
44% (76th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: CD63/ CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.14-1.19 
Show all info
Study aim
Biomarker/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDAMB231
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
10
Lowest density fraction
5%
Highest density fraction
50%
Orientation
Top-down
Speed (g)
210000
Duration (min)
960
Fraction volume (mL)
0.45
Fraction processing
Centrifugation
Pelleting: duration (min)
60
Pelleting: speed (g)
200000
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
CD63/ CD9
Flow cytometry specific beads
Antibody details provided?
No
Detected EV-associated proteins
CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
78.36
NTA
Report type
Not determined
EV concentration
Yes
EM
EM-type
Transmission­-EM
Image type
Wide-field
Report size (nm)
100
EV210490 2/5 Homo sapiens Cell culture supernatant (d)(U)C Bosque A 2016 44%

Study summary

Full title
All authors
Bosque A, Dietz L, Gallego-Lleyda A, Sanclemente M, Iturralde M, Naval J, Alava MA, Martínez-Lostao L, Thierse HJ, Anel A
Journal
Oncotarget
Abstract
We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside (show more...)We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion. (hide)
EV-METRIC
44% (76th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
PHA
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
Protein markers
EV: HSP70/ HSP90/ Hsp60/ Hsp40/ Rab5/ / VCP/ Apo2L/TRAIL/ FasL/ Lck/ alpha-Tubulin/ beta-Actin/ TCP-1alpha
non-EV: Grp94
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Jurkat
EV-harvesting Medium
Serum free medium
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 40 Ti
Pelleting: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP90/ VCP/ Apo2L/TRAIL/ FasL/ Lck/ alpha-Tubulin/ beta-Actin/ TCP-1alpha/ HSP70
Not detected EV-associated proteins
Hsp60/ Hsp40/ Rab5
Not detected contaminants
Grp94
Proteomics database
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
None
EV210490 4/5 Homo sapiens Cell culture supernatant (d)(U)C Bosque A 2016 44%

Study summary

Full title
All authors
Bosque A, Dietz L, Gallego-Lleyda A, Sanclemente M, Iturralde M, Naval J, Alava MA, Martínez-Lostao L, Thierse HJ, Anel A
Journal
Oncotarget
Abstract
We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside (show more...)We have previously characterized that FasL and Apo2L/TRAIL are stored in their bioactive form inside human T cell blasts in intraluminal vesicles present in multivesicular bodies. These vesicles are rapidly released to the supernatant in the form of exosomes upon re-activation of T cells. In this study we have compared for the first time proteomics of exosomes produced by normal human T cell blasts with those produced by tumoral Jurkat cells, with the objective of identify proteins associated with tumoral exosomes that could have a previously unrecognized role in malignancy. We have identified 359 and 418 proteins in exosomes from T cell blasts and Jurkat cells, respectively. Interestingly, only 145 (around a 40%) are common. The major proteins in both cases are actin and tubulin isoforms and the common interaction nodes correspond to these cytoskeleton and related proteins, as well as to ribosomal and mRNA granule proteins. We detected 14 membrane proteins that were especially enriched in exosomes from Jurkat cells as compared with T cell blasts. The most abundant of these proteins was valosin-containing protein (VCP), a membrane ATPase involved in ER homeostasis and ubiquitination. In this work, we also show that leukemic cells are more sensitive to cell death induced by the VCP inhibitor DBeQ than normal T cells. Furthermore, VCP inhibition prevents functional exosome secretion only in Jurkat cells, but not in T cell blasts. These results suggest VCP targeting as a new selective pathway to exploit in cancer treatment to prevent tumoral exosome secretion. (hide)
EV-METRIC
44% (76th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
PHA
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
Protein markers
EV: VCP/ alpha-Tubulin/ beta-Actin/ TCP-1alpha/ HSP90/ CD63/ HSP70
non-EV: Grp94
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
T-cell blasts
EV-harvesting Medium
Serum free medium
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 40 Ti
Pelleting: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
VCP/ alpha-Tubulin/ beta-Actin/ TCP-1alpha/ CD63/ HSP90/ HSP70
Not detected contaminants
Grp94
Proteomics database
No
Characterization: Lipid analysis
Yes
EM
EM-type
Immuno-EM
EM protein
Other/ FasL/ APO2/TRAIL
Image type
Close-up
Report size (nm)
100-130
EV210444 1/2 Homo sapiens NA (d)(U)C San Lucas FA 2016 44%

Study summary

Full title
All authors
San Lucas FA, Allenson K, Bernard V, Castillo J, Kim DU, Ellis K, Ehli EA, Davies GE, Petersen JL, Li D, Wolff R, Katz M, Varadhachary G, Wistuba I, Maitra A, Alvarez H
Journal
Ann Oncol
Abstract
The ability to perform comprehensive profiling of cancers at high resolution is essential for precis (show more...)The ability to perform comprehensive profiling of cancers at high resolution is essential for precision medicine. Liquid biopsies using shed exosomes provide high-quality nucleic acids to obtain molecular characterization, which may be especially useful for visceral cancers that are not amenable to routine biopsies. (hide)
EV-METRIC
44% (58th 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
Pancreaticobiliary cancer
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
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: speed (g)
154000
Wash: volume per pellet (ml)
70
Wash: time (min)
120
Wash: speed (g)
154000
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ HSP70/ CD81
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD63
Flow cytometry specific beads
Antibody details provided?
Yes
Selected surface protein(s)
CD63
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Close-up, Wide-field
EV210322 1/5 Homo sapiens Cell culture supernatant (d)(U)C Kibria G 2016 44%

Study summary

Full title
All authors
Kibria G, Ramos EK, Lee KE, Bedoyan S, Huang S, Samaeekia R, Athman JJ, Harding CV, Lötvall J, Harris L, Thompson CL, Liu H
Journal
Sci Rep
Abstract
Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human di (show more...)Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30-150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids. (hide)
EV-METRIC
44% (76th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
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
Protein markers
EV: CD63/ beta-actin/ CD44/ LAMP2B/ CD47
non-EV: Grp94
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDAMB231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
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)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
100000