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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
Experiment number
  • Experiments differ in Sample condition
Experiment number
  • Experiments differ in Sample condition
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV200058 1/1 Homo sapiens Cell culture supernatant (d)(U)C
DG
Luis A Arteaga-Blanco 2020 100%

Study summary

Full title
All authors
Luis A Arteaga-Blanco, Andrés Mojoli, Robson Q Monteiro, Vanessa Sandim, Rubem F S Menna-Barreto, Filipe Santos Pereira-Dutra, Patrícia T Bozza, Rafael de Oliveira Resende, Dumith Chequer Bou-Habib
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of t (show more...)Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
Other / Small EVs
Separation protocol
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
(d)(U)C + DG
Protein markers
EV: Alix/ CD81/ CD63/ beta actin
non-EV: Calnexin/ Cytochrome c
Proteomics
no
EV density (g/ml)
1.117- 1.181
Show all info
Study aim
Biomarker/protocol adaptation for the isolation and characterization of MDM-derived small EVs
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary human macrophages derived from circulating monocytes
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
130
Wash: volume per pellet (ml)
10
Wash: time (min)
70
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
130 000
Density gradient
Only used for validation of main results
Yes
Density medium
Sucrose
Type
Continuous
Lowest density fraction
10%
Highest density fraction
90%
Total gradient volume, incl. sample (mL)
11
Sample volume (mL)
0,2
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: duration (min)
70
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
130
Pelleting-wash: volume per pellet (mL)
10
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
SW 41 Ti
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD63/ beta actin/ Alix/ CD81
Not detected contaminants
Calnexin/ Cytochrome c
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
40-150
EV concentration
Yes
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
Report size (nm)
80
EV190105 1/2 Homo sapiens Cell culture supernatant DG
(d)(U)C
Emanuela Mensà 2020 100%

Study summary

Full title
All authors
Emanuela Mensà, Michele Guescini, Angelica Giuliani, Maria Giulia Bacalini, Deborah Ramini, Giacomo Corleone, Manuela Ferracin, Gianluca Fulgenzi, Laura Graciotti, Francesco Prattichizzo, Leonardo Sorci, Michela Battistelli, Vladia Monsurrò, Anna Rita Bonfigli, Maurizio Cardelli, Rina Recchioni, Fiorella Marcheselli, Silvia Latini, Serena Maggio, Mirco Fanelli, Stefano Amatori, Gianluca Storci, Antonio Ceriello, Vilberto Stocchi, Maria De Luca, Luca Magnani, Maria Rita Rippo, Antonio Domenico Procopio, Claudia Sala, Iva Budimir, Cristian Bassi, Massimo Negrini, Paolo Garagnani, Claudio Franceschi, Jacopo Sabbatinelli, Massimiliano Bonafè, Fabiola Olivieri
Journal
J Extracell Vesicles
Abstract
The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. H (show more...)The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40–100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C
Protein markers
EV: TSG101/ Calnexin/ CD63
non-EV: Albumin
Proteomics
no
EV density (g/ml)
1.08-1.10
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Human umbilical vein endothelial cells (HUVECs)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 28
Pelleting: speed (g)
103745
Wash: volume per pellet (ml)
3
Wash: time (min)
70
Wash: Rotor Type
Type 90 Ti
Wash: speed (g)
109354
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.5
Orientation
Bottom-up
Rotor type
SW 28
Speed (g)
103745
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
3.5
Pelleting: duration (min)
70
Pelleting: rotor type
Type 90 Ti
Pelleting: speed (g)
109354
Pelleting-wash: volume per pellet (mL)
3
Pelleting-wash: duration (min)
70
Pelleting-wash: speed (g)
Type 90 Ti
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ Calnexin
Not detected contaminants
Albumin
Characterization: RNA analysis
Proteinase treatment
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
20
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
80 (sEVs) / 120 (lEVs)
EV concentration
Yes
Particle yield
Number of particles per cell: 3E4 (sEVs)/1.5E4 (lEVs)
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190103 1/1 Homo sapiens Cell culture supernatant DG
(d)(U)C
Dynabeads CD9, CD63, CD81
Xiaoju Zhou 2020 100%

Study summary

Full title
All authors
Xiaoju Zhou, Brooke A Brown, Amanda P Siegel, Mohamed El Masry, Xuyao Zeng, Woran Song, Amitava Das, Puneet Khandelwal, Andrew Clark, Kanhaiya Singh, Poornachander R Guda, Mahadeo Gorain, Lava Timsina, Yi Xuan, Stephen C Jacobson, Milos V Novotny, Sashwati Roy, Mangilal Agarwal, Robert J Lee, Chandan K Sen, David E Clemmer, Subhadip Ghatak
Journal
ACS Nano
Abstract
Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a (show more...)Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP reporter (Exoκ-GFP) using tissue nanotransfection and were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low base pair RNA and were selectively engulfed by wound-macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFP in vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, TLNPκ/si-hnRNPA2B1 treated group showed significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP are recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties post-injury. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + Dynabeads CD9, CD63, CD81
Protein markers
EV: TSG101/ Alix
non-EV:
Proteomics
no
EV density (g/ml)
1.16
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HaCaT
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-120.2
Pelleting: speed (g)
245000
Wash: volume per pellet (ml)
0.5
Wash: time (min)
120
Wash: Rotor Type
TLA-120.2
Wash: speed (g)
245000
Density gradient
Only used for validation of main results
Yes
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
1.05
Sample volume (mL)
0.05
Orientation
Top-down
Rotor type
TLA-120.2
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
0.1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
120
Pelleting: rotor type
TLA-120.2
Pelleting: speed (g)
245000
Commercial kit
Dynabeads CD9, CD63, CD81
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
TSG101/ Alix/ FLOT1/ HSP90
Detected contaminants
Prohibitin
Not detected contaminants
GM130
Flow cytometry specific beads
Detected EV-associated proteins
TSG101
Other 1
Fluorescent anisotropy
Detected EV-associated proteins
TSG101
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
102
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 3.20E+07
EM
EM-type
Scanning-EM
Image type
Wide-field
EV190064 5/10 Homo sapiens Urine DG
(d)(U)C
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + UF
Protein markers
EV: TSG101/ Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101/ Alix
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
196.5
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 6/10 Homo sapiens Urine DG
(d)(U)C
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + UF
Protein markers
EV: TSG101/ Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101/ Alix
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
131.7
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 7/10 Homo sapiens Urine DG
(d)(U)C
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Prostate Cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + UF
Protein markers
EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1
non-EV: Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Prostate Cancer
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9
Detected contaminants
Tamm-Horsfall protein
Proteomics database
Yes:
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 8/10 Homo sapiens Urine DG
(d)(U)C
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
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
Bladder Cancer
Focus vesicles
extracellular vesicle
Separation protocol
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
DG + (d)(U)C + UF
Protein markers
EV: Alix/ Flotillin1/ CD9/ Syntenin-1
non-EV: Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Bladder Cancer
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Syntenin-1/ CD9
Detected contaminants
Tamm-Horsfall protein
Proteomics database
Yes:
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 9/10 Homo sapiens Urine DG
(d)(U)C
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (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
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
Renal Cancer
Focus vesicles
extracellular vesicle
Separation protocol
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
DG + (d)(U)C + UF
Protein markers
EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1
non-EV: Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Renal Cancer
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9
Detected contaminants
Tamm-Horsfall protein
Proteomics database
Yes:
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-300
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190064 10/10 Homo sapiens Tissue DG
(d)(U)C
UF
Dhondt B 2020 100%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
100% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Tissue
Sample origin
Prostate Cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + UF
Protein markers
EV: Alix/ TSG101/ Flotillin1/ CD9/ Syntenin-1
non-EV: Tamm-Horsfall protein
Proteomics
yes
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Tissue
Sample Condition
Prostate Cancer
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Syntenin-1/ TSG101/ CD9
Detected contaminants
Tamm-Horsfall protein
Proteomics database
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150.3
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
30-150
EV190044 11/11 Homo sapiens Blood plasma DG
(d)(U)C
qEV
Driedonks, Tom A.P. 2020 100%

Study summary

Full title
All authors
Tom A.P. Driedonks, Sanne Mol, Sanne de Bruin, Anna-Linda Peters, Xiaogang Zhang, Marthe F.S. Lindenbergh, Boukje M. Beuger, Anne-Marieke D. van Stalborch, Thom Spaan, Esther C. de Jong, Erhard van der Vries, Coert Margadant, Robin van Bruggen, Alexander P.J. Vlaar, Tom Groot Kormelink, and Esther N.M. Nolte-‘T Hoen
Journal
J Extracell Vesicles
Abstract
Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood p (show more...)Major efforts are made to characterize the presence of microRNA (miRNA) and messenger RNA in blood plasma to discover novel disease-associated biomarkers. MiRNAs in plasma are associated to several types of macromolecular structures, including extracellular vesicles (EV), lipoprotein particles (LPP) and ribonucleoprotein particles (RNP). RNAs in these complexes are recovered at variable efficiency by commonly used EV- and RNA isolation methods, which causes biases and inconsistencies in miRNA quantitation. Besides miRNAs, various other non-coding RNA species are contained in EV and present within the pool of plasma extracellular RNA. Members of the Y-RNA family have been detected in EV from various cell types and are among the most abundant non-coding RNA types in plasma. We previously showed that shuttling of full-length Y-RNA into EV released by immune cells is modulated by microbial stimulation. This indicated that Y-RNAs could contribute to the functional properties of EV in immune cell communication and that EV-associated Y-RNAs could have biomarker potential in immune-related diseases. Here, we investigated which macromolecular structures in plasma contain full length Y-RNA and whether the levels of three Y-RNA subtypes in plasma (Y1, Y3 and Y4) change during systemic inflammation. Our data indicate that the majority of full length Y-RNA in plasma is stably associated to EV. Moreover, we discovered that EV from different blood-related cell types contain cell-type-specific Y-RNA subtype ratios. Using a human model for systemic inflammation, we show that the neutrophil-specific Y4/Y3 ratios and PBMC-specific Y3/Y1 ratios were significantly altered after induction of inflammation. The plasma Y-RNA ratios strongly correlated with the number and type of immune cells during systemic inflammation. Cell-type-specific “Y-RNA signatures” in plasma EV can be determined without prior enrichment for EV, and may be further explored as simple and fast test for diagnosis of inflammatory responses or other immune-related diseases. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + qEV
Protein markers
EV: CD81/ CD63/ Flotillin1/ CD9
non-EV: ApoAl/ ApoB100
Proteomics
no
EV density (g/ml)
1.11 - 1.18
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4 M
Highest density fraction
2.5 M
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
0.05
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ CD81
Not detected contaminants
ApoAl/ ApoB100
Characterization: RNA analysis
Proteinase treatment
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
0.045
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.16
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
130
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200065 1/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Victoria Stary 2020 78%

Study summary

Full title
All authors
Victoria Stary, Brigitte Wolf, Daniela Unterleuthner, Julia List, Merjem Talic, Johannes Längle, Andrea Beer, Johanna Strobl, Georg Stary, Helmut Dolznig, Michael Bergmann Md
Journal
Methods & Clinical Development
Abstract
Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumo (show more...)Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies. (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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Protein markers
EV: CD9/ CD81/ TSG101
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
DLD1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
T-1250
Pelleting: speed (g)
243836
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
92,500
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD81/ TSG101
Not detected contaminants
Calreticulin
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
132
EV concentration
Yes
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
EV200065 2/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Victoria Stary 2020 78%

Study summary

Full title
All authors
Victoria Stary, Brigitte Wolf, Daniela Unterleuthner, Julia List, Merjem Talic, Johannes Längle, Andrea Beer, Johanna Strobl, Georg Stary, Helmut Dolznig, Michael Bergmann Md
Journal
Methods & Clinical Development
Abstract
Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumo (show more...)Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies. (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
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
gamma irradiation
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C + Filtration
Protein markers
EV: CD9/ CD81/ TSG101
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
gamma irradiation
EV-producing cells
DLD1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
T-1250
Pelleting: speed (g)
243836
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
92,500
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD81/ TSG101
Not detected contaminants
Calreticulin
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
137
EV concentration
Yes
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
EV200065 3/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Victoria Stary 2020 78%

Study summary

Full title
All authors
Victoria Stary, Brigitte Wolf, Daniela Unterleuthner, Julia List, Merjem Talic, Johannes Längle, Andrea Beer, Johanna Strobl, Georg Stary, Helmut Dolznig, Michael Bergmann Md
Journal
Methods & Clinical Development
Abstract
Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumo (show more...)Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies. (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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Protein markers
EV: CD9/ CD81/ TSG101
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HCT116
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
T-1250
Pelleting: speed (g)
243836
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
92,500
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD81/ TSG101
Not detected contaminants
Calreticulin
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
157
EV concentration
Yes
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
EV200065 4/4 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Victoria Stary 2020 78%

Study summary

Full title
All authors
Victoria Stary, Brigitte Wolf, Daniela Unterleuthner, Julia List, Merjem Talic, Johannes Längle, Andrea Beer, Johanna Strobl, Georg Stary, Helmut Dolznig, Michael Bergmann Md
Journal
Methods & Clinical Development
Abstract
Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumo (show more...)Background: Tumor-associated macrophages (TAM) constitute the most abundant immune cells in the tumor stroma initiating pro-inflammatory (M1) or immunosuppressive (M2) responses depending on their polarization status. Advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patients. Methods: To test the effects of radiotherapy on TAM, we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry. We furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short-course radiotherapy and compared findings to non-pretreated rectal cancer using an immunostaining approach. Organotypic assays (OTA) consisting of macrophages, cancer-associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophages. Results: We demonstrate that short-course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of TAM towards an M1-like pro-inflammatory phenotype. In addition, ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for T-cell activation. In OTA we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles (EV) derived from irradiated tumor cells. We identified high mobility group box 1 in EV from irradiated tumor cells as a potential effector signal in that crosstalk. Conclusions: Our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro-inflammation. Our data indicate that clinically applied short-term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategies. (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
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
gamma irradiation
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C + Filtration
Protein markers
EV: CD9/ CD81/ TSG101
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
gamma irradiation
EV-producing cells
HCT116
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Between 50,000 g and 100,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
T-1250
Pelleting: speed (g)
243836
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
92,500
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD81/ TSG101
Not detected contaminants
Calreticulin
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
161
EV concentration
Yes
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
EV200036 1/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
Commercial method
Juan Antonio Fafián-Labora 2020 78%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
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
Young donors
Focus vesicles
extracellular vesicle
Separation protocol
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
DG + (d)(U)C + Commercial method
Protein markers
EV: Alix/ TSG101/ GSTM2
non-EV: Calnexin/ Actin-beta
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Young donors
EV-producing cells
human skin primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ GSTM2
Not detected contaminants
Calnexin/ Actin-beta
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 3/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
Commercial method
Juan Antonio Fafián-Labora 2020 78%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (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
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
Old donors
Focus vesicles
extracellular vesicle
Separation protocol
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
DG + (d)(U)C + Commercial method
Protein markers
EV: Alix/ TSG101/ GSTM2
non-EV: Calnexin/ Actin-beta
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Old donors
EV-producing cells
human skin primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ GSTM2
Not detected contaminants
Calnexin/ Actin-beta
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV190084 1/2 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Greet Merckx 2020 78%

Study summary

Full title
All authors
Greet Merckx, Baharak Hosseinkhani, Sören Kuypers, Sarah Deville, Joy Irobi, Inge Nelissen, Luc Michiels, Ivo Lambrichts, Annelies Bronckaers
Journal
Cells
Abstract
Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marr (show more...)Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis. (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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Protein markers
EV: CD9/ CD63/ ANXA2/ CD81
non-EV: Bax
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Dental pulp stromal cells
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ ANXA2/ CD81
Not detected contaminants
Bax
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190084 2/2 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Greet Merckx 2020 78%

Study summary

Full title
All authors
Greet Merckx, Baharak Hosseinkhani, Sören Kuypers, Sarah Deville, Joy Irobi, Inge Nelissen, Luc Michiels, Ivo Lambrichts, Annelies Bronckaers
Journal
Cells
Abstract
Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marr (show more...)Blood vessel formation or angiogenesis is a key process for successful tooth regeneration. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) possess paracrine proangiogenic properties, which are, at least partially, induced by their extracellular vesicles (EVs). However, the isolation of BM-MSCs is associated with several drawbacks, which could be overcome by MSC-like cells of the teeth, called dental pulp stromal cells (DPSCs). This study aims to compare the angiogenic content and functions of DPSC and BM-MSC EVs and conditioned medium (CM). The angiogenic protein profile of DPSC- and BM-MSC-derived EVs, CM and EV-depleted CM was screened by an antibody array and confirmed by ELISA. Functional angiogenic effects were tested in transwell migration and chicken chorioallantoic membrane assays. All secretion fractions contained several pro- and anti-angiogenic proteins and induced in vitro endothelial cell motility. This chemotactic potential was higher for (EV-depleted) CM, compared to EVs with a stronger effect for BM-MSCs. Finally, BM-MSC CM, but not DPSC CM, nor EVs, increased in ovo angiogenesis. In conclusion, we showed that DPSCs are less potent in relation to endothelial cell chemotaxis and in ovo neovascularization, compared to BM-MSCs, which emphasizes the importance of choice of cell type and secretion fraction for stem cell-based regenerative therapies in inducing angiogenesis. (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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Protein markers
EV: CD9/ CD63/ ANXA2/ CD81
non-EV: Bax
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Bone marrow derived mesenchymal stromal cells
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ ANXA2/ CD81
Not detected contaminants
Bax
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190079 1/2 Homo sapiens kidney tissue supernatant (d)(U)C
Filtration
Zieren RC 2020 78%

Study summary

Full title
All authors
Zieren RC, Dong L, Pierorazio PM, Pienta KJ, de Reijke TM, Amend SR.
Journal
Med Oncol
Abstract
Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive bio (show more...)Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive biomarkers are needed to aid diagnosis and treatment. Extracellular vesicles (EVs), membranous vesicles secreted by all cells, are a promising potential source for cancer biomarkers, but new methods are required that are both sensitive and specific for cancer identification. We have developed an EV isolation protocol optimized for kidney tumor and normal kidney tissue that yields a high vesicle concentration, confirmed by nanoparticle tracking analysis (NanoSight) and by nanoscale flow cytometry (NanoFCM). Using Western blot, we confirmed presence of EV markers CD81, CD63, flotillin-1, and absence of cellular debris, calnexin. Transmission electron microscopy images demonstrate intact membranous EVs. This new method improves existing protocols with additional steps to reduce contaminants in the EV product. Characterization of our isolation product confirms successful isolation of EVs with minimal contamination. The particle yields of our protocol are consistent and high as assessed by both standard and novel methods. This optimized protocol will contribute to biomarker discovery and biological studies of EVs in renal cancer. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
kidney tissue 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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + Filtration
Protein markers
EV: CD81/ CD63/ Flotillin1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
kidney tissue supernatant
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Filtration steps
> 0.45 µm, 0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
163
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Instrument was manufactured for small EVs
Calibration bead size
200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report type
Modus
Report size
57
EV-concentration
Yes
EV190079 2/2 Homo sapiens kidney tissue supernatant (d)(U)C
Filtration
Zieren RC 2020 78%

Study summary

Full title
All authors
Zieren RC, Dong L, Pierorazio PM, Pienta KJ, de Reijke TM, Amend SR.
Journal
Med Oncol
Abstract
Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive bio (show more...)Renal cell carcinoma is a lethal disease that is often discovered incidentally. New non-invasive biomarkers are needed to aid diagnosis and treatment. Extracellular vesicles (EVs), membranous vesicles secreted by all cells, are a promising potential source for cancer biomarkers, but new methods are required that are both sensitive and specific for cancer identification. We have developed an EV isolation protocol optimized for kidney tumor and normal kidney tissue that yields a high vesicle concentration, confirmed by nanoparticle tracking analysis (NanoSight) and by nanoscale flow cytometry (NanoFCM). Using Western blot, we confirmed presence of EV markers CD81, CD63, flotillin-1, and absence of cellular debris, calnexin. Transmission electron microscopy images demonstrate intact membranous EVs. This new method improves existing protocols with additional steps to reduce contaminants in the EV product. Characterization of our isolation product confirms successful isolation of EVs with minimal contamination. The particle yields of our protocol are consistent and high as assessed by both standard and novel methods. This optimized protocol will contribute to biomarker discovery and biological studies of EVs in renal cancer. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
kidney tissue supernatant
Sample origin
kidney cancer
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C + Filtration
Protein markers
EV: CD81/ CD63/ Flotillin1
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
kidney tissue supernatant
Sample Condition
kidney cancer
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
30
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Filtration steps
> 0.45 µm, 0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
133
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Instrument was manufactured for small EVs
Calibration bead size
200
Report type
Modus
Reported size (nm)
57
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report type
Modus
Report size
57
EV-concentration
Yes
EV190076 1/1 Homo sapiens Urine (d)(U)C Musante L 2020 78%

Study summary

Full title
All authors
Musante L, Bontha SV, La Salvia S, Fernandez-Piñeros A, Lannigan J, Le TH, Mas V, Erdbrügger U
Journal
Sci Rep
Abstract
Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centri (show more...)Urinary extracellular vesicles (uEVs) provide bio-markers for kidney and urogenital diseases. Centrifugation is the most common method used to enrich uEVs. However, a majority of studies to date have focused on the ultracentrifugation pellet, potentially losing a novel source of important biomarkers that could be obtained at lower centrifugation. Thus, the aim of this study is to rigorously characterize for the first time uEVs in the low speed pellet and determine the minimal volume of urine required for proteomic analysis (≥9.0 mL urine) and gene ontology classification identified 75% of the protein as extracellular exosomes. Cryo-Transmission Electron Microscopy (≥3.0 mL urine) provided evidence of a heterogeneous population of EVs for size and morphology independent of uromodulin filaments. Western blot detected several specific uEV kidney and EV markers (≥4.5 mL urine per lane). microRNAs quantification by qPCR was possible with urine volume as low as 0.5 mL. Particle enumeration with tunable resistive pulse sensing, nano particles tracking analysis and single EV high throughput imaging flow cytometry are possible starting from 0.5 and 3.0 mL of urine respectively. This work characterizes a neglected source of uEVs and provides guidance with regard to volume of urine necessary to carry out multi-omic studies and reveals novel aspects of uEV analysis such as autofluorescence of podocyte origin. (hide)
EV-METRIC
78% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C
Protein markers
EV: TSG101/ Podocin/ Podocalyxin/ Collectrin/ IGFBP7/ CD9
non-EV: Calnexin/ Tamm-Horsfall protein/ Albumin/ Calreticulin
Proteomics
yes
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
FA-45-24-11
Pelleting: speed (g)
21130
Wash: volume per pellet (ml)
1.2
Wash: time (min)
30
Wash: Rotor Type
FA-45-24-11
Wash: speed (g)
21130
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD9/ Podocalyxin/ Collectrin/ Podocin/ TSG101
Detected contaminants
Calnexin/ Calreticulin/ Albumin/ Tamm-Horsfall protein
Flow cytometry
Type of Flow cytometry
ImageStreamX Mark II
Hardware adjustments
Imaging flow cytometry (IFCM) is a method combining flow cytometry with imaging. All signals are collected through microscope objectives and quantified based on images detected by charge coupled devic
Detected EV-associated proteins
Podocalyxin/ Collectrin/ IGFBP7
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
175
EV concentration
Yes
TRPS
Report type
Modus
Reported size (nm)
173
EV concentration
Yes
EM
EM-type
Cryo-EM
Image type
Close-up, Wide-field
EV190072 1/4 Homo sapiens Cell culture supernatant (d)(U)C
IAF
Hosseinkhani, Baharak 2020 78%

Study summary

Full title
All authors
Baharak Hosseinkhani, Nynke M S van den Akker, Daniel G M Molin, Luc Michiels
Journal
J Extracell Vesicles
Abstract
Substantial research has been devoted to discovering the translational potential of extracellular ve (show more...)Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization. (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
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
TNF-treated
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C + IAF
Protein markers
EV: / ANXA2/ CD63/ CD9/ ICAM1
non-EV: BAX
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
TNF-treated
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
Wash: time (min)
Wash: Rotor Type
Wash: speed (g)
EV-subtype
Used subtypes
ICAM1 positive
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ ICAM1/ ANXA2
Not detected contaminants
BAX
ELISA
Detected EV-associated proteins
ICAM1
Other 1
Inflammation array C3
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190072 2/4 Homo sapiens Cell culture supernatant (d)(U)C
IAF
Hosseinkhani, Baharak 2020 78%

Study summary

Full title
All authors
Baharak Hosseinkhani, Nynke M S van den Akker, Daniel G M Molin, Luc Michiels
Journal
J Extracell Vesicles
Abstract
Substantial research has been devoted to discovering the translational potential of extracellular ve (show more...)Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization. (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
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
TNF-treated
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C + IAF
Protein markers
EV: / ANXA2/ CD63/ CD9/ ICAM1
non-EV: BAX
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
TNF-treated
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
Wash: time (min)
Wash: Rotor Type
Wash: speed (g)
EV-subtype
Used subtypes
ICAM1 negative
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Not detected EV-associated proteins
CD9/ CD63/ ICAM1/ ANXA2
Not detected contaminants
BAX
ELISA
Not detected EV-associated proteins
Not detected contaminants
ICAM1
Other 1
Inflammation array C3
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-100
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190072 3/4 Homo sapiens Cell culture supernatant (d)(U)C
IAF
Hosseinkhani, Baharak 2020 78%

Study summary

Full title
All authors
Baharak Hosseinkhani, Nynke M S van den Akker, Daniel G M Molin, Luc Michiels
Journal
J Extracell Vesicles
Abstract
Substantial research has been devoted to discovering the translational potential of extracellular ve (show more...)Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization. (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
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
TNF-treated
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C + IAF
Protein markers
EV: / ANXA2/ CD63/ CD9/ ICAM1
non-EV: BAX
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
TNF-treated
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
S-4-72
Pelleting: speed (g)
10000
Wash: volume per pellet (ml)
Wash: time (min)
Wash: Rotor Type
Wash: speed (g)
EV-subtype
Used subtypes
ICAM1 positive
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ ANXA2/ ICAM1
Not detected EV-associated proteins
Not detected contaminants
BAX
ELISA
Detected EV-associated proteins
ICAM1
Other 1
Inflammation array C3
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-400
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190072 4/4 Homo sapiens Cell culture supernatant (d)(U)C
IAF
Hosseinkhani, Baharak 2020 78%

Study summary

Full title
All authors
Baharak Hosseinkhani, Nynke M S van den Akker, Daniel G M Molin, Luc Michiels
Journal
J Extracell Vesicles
Abstract
Substantial research has been devoted to discovering the translational potential of extracellular ve (show more...)Substantial research has been devoted to discovering the translational potential of extracellular vesicles (EV) as a reliable liquid biopsy in the diagnosis and monitoring of several life-affecting diseases, including chronic inflammatory diseases (CID). So far, the role of EV in the development of CID remains largely unknown due to the lack of specific tools to separate the disease-associated EV subtypes. Therefore, this study aims to fractionate inflammation-associated EV (sub)populations using a two-step separation strategy based on their size combined with a specific inflammatory marker (ICAM-1) and to unravel their proteome signature and functional integrity at the onset of vascular inflammation. Here, we report that vascular endothelial cells upon inflammation release two heterogeneous size-based populations of EV (EV-10 K and EV-110 K) sharing a cocktail of inflammatory proteins, chemokines, and cytokines (chiefly: ICAM-1, CCL-2, CCL-4, CCL-5, IL-8 and CXCL-10). The co-enrichment of ICAM-1 and classical EV markers within these two size-based populations gave us a promising opportunity to further separate the inflammation-associated EV subpopulations, using an immuno-affinity methodology. Protein profiling of EV subpopulations highlighted that the phenotypic state of inflamed endothelial cells is preferentially mirrored in secreted medium- and large-sized ICAM-1 (+) EV. As functional players, the smaller-sized EV and especially their ICAM-1 (+) EV subpopulation promote the migration of THP-1 monocytes, whereas the large ICAM-1 (+) EV were more potent to induce ICAM-1 expression in recipient endothelial cells. This study provides new insights into the immunomodulatory content of inflammation-associated EV (sub)populations and their functional contributions to the initiation of vascular inflammation (ICAM-1 expression) and monocyte mobilization. (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
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
TNF-treated
Focus vesicles
extracellular vesicle
Separation protocol
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
(d)(U)C + IAF
Protein markers
EV: / ANXA2/ CD63/ CD9/ ICAM1
non-EV: BAX
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
TNF-treated
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
S-4-72
Pelleting: speed (g)
10000
EV-subtype
Used subtypes
ICAM1 negative
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ ANXA2
Not detected EV-associated proteins
ICAM1
Not detected contaminants
BAX
ELISA
Not detected EV-associated proteins
Not detected contaminants
ICAM1
Other 1
Inflammation array C3
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-400
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV190064 1/10 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
UF
Dhondt B 2020 78%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (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
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
pMET7-gag-EGFP transfected
Focus vesicles
extracellular vesicle
Separation protocol
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
DG + (d)(U)C + Filtration + UF
Protein markers
EV: Flotillin1/ Syntenin-1/ gag-EGFP
non-EV:
Proteomics
no
EV density (g/ml)
1.087-1.109
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
pMET7-gag-EGFP transfected
EV-producing cells
HEK293T
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ Syntenin-1/ gag-EGFP
Fluorescent NTA
Relevant measurements variables specified?
NA
Detected EV-associated proteins
gag-EGFP
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Immuno-EM
Proteïns
CD63
Image type
Close-up, Wide-field
EV190022 1/1 Bos taurus Bovine embryo culture media DG
(d)(U)C
SEC
Krishna Chaitanya Pavani 2020 78%

Study summary

Full title
All authors
Krishna Chaitanya Pavani, Xiaoyuan Lin, Joachim Hamacher, Wim Van Den Broeck, Liesbeth Couck, Luc Peelman, An Hendrix and Ann Van Soom
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst emb (show more...)Extracellular vesicles (EVs) have been identified as one of the communication mechanisms amongst embryos. They are secreted into the embryo culture medium and, as such, represent a source of novel biomarkers for identifying the quality of cells and embryos. However, only small amounts of embryo-conditioned medium are available, which represents a challenge for EV enrichment. Our aim is to assess the suitability of different EV separation methods to retrieve EVs with high specificity and sufficient efficiency. Bovine embryo-conditioned medium was subjected to differential ultracentrifugation (DU), OptiPrepTM density gradient (ODG) centrifugation, and size exclusion chromatography. Separated EVs were characterized by complementary characterization methods, including Western blot, electron microscopy, and nanoparticle tracking analysis, to assess the efficiency and specificity. OptiPrepTM density gradient centrifugation outperformed DU and SEC in terms of specificity by substantial removal of contaminating proteins such as ribonucleoprotein complexes (Argonaute-2 (AGO-2)) and lipoproteins (ApoA-I) from bovine embryo-derived EVs (density: 1.02–1.04, 1.20–1.23 g/mL, respectively). In conclusion, ODG centrifugation is the preferred method for identifying EV-enriched components and for improving our understanding of EV function in embryo quality and development. (hide)
EV-METRIC
78% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Bovine embryo culture media
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + SEC
Protein markers
EV: CD63
non-EV: Argonaute2/ APOA1
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Bovine embryo culture media
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
180
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63
Detected contaminants
APOA1/ Argonaute2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
133.8+-6.8
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
25-250
EV190006 1/2 Homo sapiens Cell culture supernatant DC
DG
(d)(U)C
Altei, Wanessa F. 2020 78%

Study summary

Full title
All authors
Wanessa F Altei, Bianca C Pachane, Patty K Dos Santos, Lígia N M Ribeiro, Bong Hwan Sung, Alissa M Weaver, Heloisa S Selistre-de-Araújo
Journal
Cell Commun Signal
Abstract
Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from (show more...)Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion: In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs. (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
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
immortalized
Focus vesicles
exosome
Separation protocol
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
DC + DG + (d)(U)C
Protein markers
EV: TSG101/ CD63/ Flotillin1
non-EV:
Proteomics
no
EV density (g/ml)
1.11
Show all info
Study aim
Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
immortalized
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
4
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
Type 40
Speed (g)
100000
Duration (min)
18
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
180
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
3
Pelleting-wash: duration (min)
180
Pelleting-wash: speed (g)
TLA-100.3
Density cushion
Density medium
Iodixanol
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63
Not detected EV-associated proteins
TSG101
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
107
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
100
BR2025GX 1/2 Mus musculus Blood plasma DG
(d)(U)C
Zheng, Xi 2020 77%

Study summary

Full title
All authors
Xi Zheng, Kailun Xu, Biting Zhou, Ting Chen, Yanqin Huang, Qilong Li, Fei Wen, Weiting Ge, Jian Wang, Shaojun Yu, Lifeng Sun, Liang Zhu, Wei Liu, Huanhuan Gao, Liang Yue, Xue Cai, Qiushi Zhang, Guan Ruan, Tiansheng Zhu, Zhicheng Wu, Yi Zhu, Yingkuan Shao, Tiannan Guo, and Shu Zheng
Journal
J Extracell Vesicles
Abstract
Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liqui (show more...)Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to low invasiveness and high reproducibility. In addition, circulating extracellular vesicles (crEVs, extracellular vesicles isolated from plasma) expressing tumour-specific proteins are potential biomarkers for various cancers. Here, we present a data-independent acquisition (DIA)-mass spectrometry (MS)-based diagnostic method for liquid biopsies. Methods: Extracellular vesicles (EVs) were isolated from culture supernatants of human CRC cell lines, and plasma of patients with CRC at different tumour stages, by overnight ultracentrifugation coupled with sucrose density gradient centrifugation. Tumour-specific EV proteins were prioritized using Tandem Mass Tag (TMT)-based shotgun proteomics and phosphoproteomics. The results were verified in a second independent cohort and a mouse tumour-bearing model using Western blotting (WB). The candidate biomarkers were further validated in a third cohort by DIA-MS. Finally, the DIA-MS methodology was accelerated to permit high-throughput detection of EV biomarkers in another independent cohort of patients with CRC and healthy controls. Results: High levels of total and phosphorylated fibronectin 1 (FN1) in crEVs, haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with cancer progression. FGA was the most dominant biomarker candidate. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crEVs were likely released by CRC cells. Furthermore, fast DIA-MS and parallel reaction monitoring (PRM)-MS both confirmed that FGA+ crEVs could distinguish colon adenoma with an area of curve (AUC) in the receiver operating characteristic (ROC) curve of 0.949 and patients with CRC (AUC of ROC is 1.000) from healthy individuals. The performance outperformed conventional tumour biomarkers. The DIA-MS quantification of FGA+ crEVs among three groups agreed with that from PRM-MS. Conclusion: DIA-MS detection of FGA+ crEVs is a potential rapid and non-invasive screening tool to identify early stage CRC. (hide)
EV-METRIC
77% (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
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, adenoma, colorectal cancer
Focus vesicles
exosome
Separation protocol
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
DG + (d)(U)C
Adj. k-factor
88.86 (pelleting) / 43.64 (washing)
Protein markers
EV: TSG101/ HSP70/ CD63
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker, Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Blood plasma
Sample Condition
Healthy, adenoma, colorectal cancer
Separation Method
Differential ultracentrifugation
centrifugation steps
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
1200
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
160000
Pelleting: adjusted k-factor
88.86
Wash: time (min)
1200
Wash: Rotor Type
MLA-130
Wash: speed (g)
160000
Wash: adjusted k-factor
43.64
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
0.25M
Highest density fraction
2M
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
150
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
150
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
142.1
Pelleting-wash: volume per pellet (mL)
1
Pelleting-wash: duration (min)
150
Pelleting-wash: rotor type
142.1
Pelleting-wash: speed (g)
MLS-50
Pelleting-wash: adjusted k-factor
142.1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101
Proteomics database
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
EM
EM-type
Transmission-EM
Image type
Wide-field
BR2025GX 2/2 Homo sapiens Blood plasma DG
(d)(U)C
Zheng, Xi 2020 77%

Study summary

Full title
All authors
Xi Zheng, Kailun Xu, Biting Zhou, Ting Chen, Yanqin Huang, Qilong Li, Fei Wen, Weiting Ge, Jian Wang, Shaojun Yu, Lifeng Sun, Liang Zhu, Wei Liu, Huanhuan Gao, Liang Yue, Xue Cai, Qiushi Zhang, Guan Ruan, Tiansheng Zhu, Zhicheng Wu, Yi Zhu, Yingkuan Shao, Tiannan Guo, and Shu Zheng
Journal
J Extracell Vesicles
Abstract
Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liqui (show more...)Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to low invasiveness and high reproducibility. In addition, circulating extracellular vesicles (crEVs, extracellular vesicles isolated from plasma) expressing tumour-specific proteins are potential biomarkers for various cancers. Here, we present a data-independent acquisition (DIA)-mass spectrometry (MS)-based diagnostic method for liquid biopsies. Methods: Extracellular vesicles (EVs) were isolated from culture supernatants of human CRC cell lines, and plasma of patients with CRC at different tumour stages, by overnight ultracentrifugation coupled with sucrose density gradient centrifugation. Tumour-specific EV proteins were prioritized using Tandem Mass Tag (TMT)-based shotgun proteomics and phosphoproteomics. The results were verified in a second independent cohort and a mouse tumour-bearing model using Western blotting (WB). The candidate biomarkers were further validated in a third cohort by DIA-MS. Finally, the DIA-MS methodology was accelerated to permit high-throughput detection of EV biomarkers in another independent cohort of patients with CRC and healthy controls. Results: High levels of total and phosphorylated fibronectin 1 (FN1) in crEVs, haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with cancer progression. FGA was the most dominant biomarker candidate. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crEVs were likely released by CRC cells. Furthermore, fast DIA-MS and parallel reaction monitoring (PRM)-MS both confirmed that FGA+ crEVs could distinguish colon adenoma with an area of curve (AUC) in the receiver operating characteristic (ROC) curve of 0.949 and patients with CRC (AUC of ROC is 1.000) from healthy individuals. The performance outperformed conventional tumour biomarkers. The DIA-MS quantification of FGA+ crEVs among three groups agreed with that from PRM-MS. Conclusion: DIA-MS detection of FGA+ crEVs is a potential rapid and non-invasive screening tool to identify early stage CRC. (hide)
EV-METRIC
77% (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
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, adenoma, colorectal cancer
Focus vesicles
exosome
Separation protocol
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
DG + (d)(U)C
Adj. k-factor
88.86 (pelleting) / 43.64 (washing)
Protein markers
EV: TSG101/ HSP70/ CD63
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Healthy, adenoma, colorectal cancer
Separation Method
Differential ultracentrifugation
centrifugation steps
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
1200
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
160000
Pelleting: adjusted k-factor
88.86
Wash: time (min)
1200
Wash: Rotor Type
MLA-130
Wash: speed (g)
160000
Wash: adjusted k-factor
43.64
Density gradient
Density medium
Sucrose
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
0.25M
Highest density fraction
2M
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
150
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
150
Pelleting: rotor type
MLS-50
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
142.1
Pelleting-wash: volume per pellet (mL)
1
Pelleting-wash: duration (min)
150
Pelleting-wash: rotor type
142.1
Pelleting-wash: speed (g)
MLS-50
Pelleting-wash: adjusted k-factor
142.1
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101
Proteomics database
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200042 1/2 Mus musculus Cell culture supernatant DG
(d)(U)C
DC
Filtration
Laura Bouchareychas 2020 75%

Study summary

Full title
All authors
Laura Bouchareychas, Phat Duong, Sergio Covarrubias, Eric Alsop, Tuan Anh Phu, Allen Chung, Michael Gomes, David Wong, Bessie Meechoovet, Allyson Capili, Ryo Yamamoto, Hiromitsu Nakauchi, Michael T McManus, Susan Carpenter, Kendall Van Keuren-Jensen, Robert L Raffai
Journal
Cell Rep
Abstract
Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remai (show more...)Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe-/- mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery. (hide)
EV-METRIC
75% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + DC + Filtration
Protein markers
EV: Alix/ Flotillin1/ CD9
non-EV: Calnexin/ GM130
Proteomics
no
EV density (g/ml)
1.09
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Bone marrow-derived macrophages (BMDMs)
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.22µm or 0.2µm
Density cushion
Density medium
Iodixanol
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ Alix
Not detected contaminants
Calnexin/ GM130
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
Other;RNase A/T1 Mix
RNAse concentration
0.4
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
75.66
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 2.70E+09
EV190077 1/4 Bos taurus Milk DG
(d)(U)C
milk acidification
casein removal
SEC
Andrea Ridolfi 2020 75%

Study summary

Full title
All authors
Andrea Ridolfi, Marco Brucale, Costanza Montis, Lucrezia Caselli, Lucia Paolini, Anne Borup, Anders T Boysen, Francesca Loria, Martijn J C van Herwijnen, Marije Kleinjan, Peter Nejsum, Natasa Zarovni, Marca H M Wauben, Debora Berti, Paolo Bergese, Francesco Valle
Journal
Anal Chem
Abstract
The mechanical properties of extracellular vesicles (EVs) are known to influence their biological fu (show more...)The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role. (hide)
EV-METRIC
75% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + milk acidification + casein removal + SEC
Protein markers
EV: TSG101/ CD63/ Flotillin1/ CD9/ MFGE8
non-EV: beta-lactoglobulin/ Casein
Proteomics
no
EV density (g/ml)
1.06-1.19
Show all info
Study aim
Other/Biophysical characterization
Sample
Species
Bos taurus
Sample Type
Milk
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
16
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
12.45
Sample volume (mL)
6.5
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
197000
Duration (min)
960
Fraction volume (mL)
0.5
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
15
Sample volume/column (mL)
2
Resin type
Sephadex G-100
Other
Name other separation method
milk acidification;casein removal
Characterization: Protein analysis
Protein Concentration Method
Other;Colorimetric Nanoplasmonic Assay
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ MFGE8/ TSG101
Detected contaminants
beta-lactoglobulin
Not detected contaminants
Casein
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
40-160
EV190064 3/10 Homo sapiens Urine (d)(U)C
SEC
SEC (non-commercial)
UF
Dhondt B 2020 75%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
75% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(d)(U)C + SEC + SEC (non-commercial) + UF
Protein markers
EV: Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Other
Name other separation method
Size-exclusion chromatography (non-commercial)
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ CD9
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-200
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
130
EV190037 1/1 Homo sapiens Blood plasma Filtration
qEV
Laetitia S. Gaspar 2020 75%

Study summary

Full title
All authors
Laetitia S. Gaspar, Magda M. Santana, Carina Henriques, Maria M. Pinto, Teresa M. Ribeiro-Rodrigues, Henrique Girão, Rui Jorge Nobre, Luís Pereira de Almeida
Journal
Methods & Clinical Development
Abstract
Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulatin (show more...)Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulating in complex biological fluids, such as plasma. RNAs are extremely specific to health and disease, being powerful tools for diagnosis, treatment response monitoring, and development of new therapeutic strategies for several diseases. In this context, EVs are potential sources of disease biomarkers and promising delivery vehicles. However, standardized and reproducible EV isolation protocols easy to implement in clinical practice are missing. Here, a size exclusion chromatography-based protocol for EV-isolation from human plasma was optimized. We propose a workflow to isolate EVs for transcriptional research that allows concomitant analysis of particle number and size, total protein, and quantification of a major plasma contaminant. This protocol yields 7.54 × 109 ± 1.22 × 108 particles, quantified by nanoparticle tracking analysis, with a mean size of 115.7 ± 11.12 nm and a mode size of 83.13 ± 4.72 nm, in a ratio of 1.19 × 1010 ± 7.38 × 109 particles/μg of protein, determined by Micro Bicinchoninic Acid (BCA) Protein Assay, and 3.09 ± 0.7 ng RNA, assessed by fluorescence-based RNA-quantitation, from only 900 μL of plasma. The protocol is fast and easy to implement and has potential for application in biomarkers research, therapeutic strategies development, and clinical practice. (hide)
EV-METRIC
75% (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
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Filtration + qEV
Protein markers
EV: TSG101/ Analysed only by WB and Dot Blot. We used ELISA to analyse the presence of contaminants/ CD63/ CD81/ GAPDH/ Anxa5/ Alix/ ICAM/ Flotillin1/ LAMP2/ EpCAM
non-EV: Calnexin/ Albumin/ GM130/ ApoB100/ ApoA1
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Filtration steps
> 0.45 µm,
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ LAMP2/ CD81
Not detected EV-associated proteins
GAPDH
Not detected contaminants
Calnexin
ELISA
Detected EV-associated proteins
Analysed only by WB and Dot Blot. We used ELISA to analyse the presence of contaminants
Detected contaminants
ApoA1/ ApoB100/ Albumin
Other 1
Dot Blot Array
Detected EV-associated proteins
Flotillin1/ CD63/ EpCAM/ ICAM/ Anxa5/ CD81/ TSG101
Not detected EV-associated proteins
Alix
Not detected contaminants
GM130
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
89.04
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200033 2/2 Indoor dust Indoor dust DG
(d)(U)C
Dissolving indoor dust in PBS
Filtration
Dinh, Nhung Thi Hong 2020 71%

Study summary

Full title
All authors
Nhung Thi Hong Dinh, Jaewook Lee, Jaemin Lee, Sang Soo Kim, Gyeongyun Go, Seoyoon Bae, Ye In Jun, Yae Jin Yoon, Tae-Young Roh, Yong Song Gho
Journal
J Extracell Vesicles
Abstract
Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust cont (show more...)Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust contains extracellular vesicles (EVs), which are associated with pulmonary inflammation. However, it has not been reported whether indoor dust EVs affect the cancer lung metastasis. In this study, we isolated indoor dust EVs and investigated their roles in cancer lung metastasis. Upon intranasal administration, indoor dust EVs enhanced mouse melanoma lung metastasis in a dose-dependent manner in mice. Pre-treatment or co-treatment of indoor dust EVs significantly promoted melanoma lung metastasis, whereas post-treatment of the EVs did not. In addition, the lung lysates from indoor dust EV-treated mice significantly increased tumour cell migration in vitro. We observed that tumour necrosis factor-α played important roles in indoor dust EV-mediated promotion of tumour cell migration in vitro and cancer lung metastasis in vivo. Furthermore, Pseudomonas EVs, the main components of indoor dust EVs, and indoor dust EVs showed comparable effects in promoting tumour cell migration in vitro and cancer lung metastasis in vivo. Taken together, our results suggest that indoor dust EVs, at least partly contributed by Pseudomonas EVs, are potential promoting agents of cancer lung metastasis. (hide)
EV-METRIC
71% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Indoor dust
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + (d)(U)C + Dissolving indoor dust in PBS + Filtration
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
N/A
Show all info
Study aim
Function
Sample
Species
Indoor dust
Sample Type
Indoor dust
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
150000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
5.25
Sample volume (mL)
2.5
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
200000
Duration (min)
120
Fraction volume (mL)
0.5
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Protein Concentration Method
Bradford
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
129.6
NTA
Report type
Not Reported
EV concentration
Yes
Particle yield
as number of particle per gram of starting sample;Yes, other: 1.10E+11
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200054 1/7 Homo sapiens Brain tissue (d)(U)C
Filtration
DC
Huang, Yiyao 2020 67%

Study summary

Full title
All authors
Yiyao Huang, Lesley Cheng, Andrey Turchinovich, Vasiliki Mahairaki, Juan C Troncoso, Olga Pletniková, Norman J Haughey, Laura J Vella, Andrew F Hill, Lei Zheng, Kenneth W Witwer
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processe (show more...)Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease. (hide)
EV-METRIC
67% (83rd percentile of all experiments on the same sample type)
 Reported
 Not