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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
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Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV180041 1/1 Mus musculus 4T1 DG
(d)(U)C
Keklikoglou I 2018 100%

Study summary

Full title
All authors
Keklikoglou I, Cianciaruso C, Güç E, Squadrito ML, Spring LM, Tazzyman S, Lambein L, Poissonnier A, Ferraro GB, Baer C, Cassará A, Guichard A, Iruela-Arispe ML, Lewis CE, Coussens LM, Bardia A, Jain RK, Pollard JW, De Palma M
Journal
Nat Cell Biol
Abstract
Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental s (show more...)Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
191 (pelleting) / 191 (washing)
Protein markers
EV: CD81/ CD9/ Syntenin-1
non-EV: Gp96
Proteomics
yes
Show all info
Study aim
Function, Biomarker, Biogenesis/cargo sorting, Mechanism of uptake/transfer, Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
4T1
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
134000
Pelleting: adjusted k-factor
191.0
Wash: time (min)
70
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
134000
Wash: adjusted k-factor
191.0
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
10
Highest density fraction
50
Sample volume (mL)
0.1
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
100000
Duration (min)
70
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: duration (min)
70
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
110000
Pelleting: adjusted k-factor
251.4
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
7.3
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD81, Syntenin-1
Not detected contaminants
Gp96
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
140
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV180007 1/1 Bos taurus Embryo culture DG
UF
Pavani KC 2018 100%

Study summary

Full title
All authors
Pavani KC, Hendrix A, Van Den Broeck W, Couck L, Szymanska K, Lin X, De Koster J, Van Soom A, Leemans B
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in vari (show more...)Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrepTM density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25⁻230 nm with an average concentration of 236.5 ± 1.27 × 10⁸ particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Embryo culture
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
UF
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: ApoA1/ Argonaute-2
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Bos taurus
Sample Type
Embryo culture
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Sample volume (mL)
1
Orientation
Top-down (sample migrates downwards)
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: speed (g)
100000
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, TSG101
Not detected contaminants
Argonaute-2, ApoA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
133.8±6.8
EV concentration
Yes
EM
EM-type
Transmission-EM/ Immune-EM
EM protein
CD63
Image type
Close-up, Wide-field
EV170030 1/1 Mus musculus primary bone marrow dendritic cells DG
(d)(U)C
Driedonks, Tom A. P. 2018 100%

Study summary

Full title
All authors
Driedonks TAP, van der Grein SG, Ariyurek Y, Buermans HPJ, Jekel H, Chow FWN, Wauben MHM, Buck AH, 't Hoen PAC, Nolte-'t Hoen ENM
Journal
Cell Mol Life Sci
Abstract
The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of inte (show more...)The release and uptake of nano-sized extracellular vesicles (EV) is a highly conserved means of intercellular communication. The molecular composition of EV, and thereby their signaling function to target cells, is regulated by cellular activation and differentiation stimuli. EV are regarded as snapshots of cells and are, therefore, in the limelight as biomarkers for disease. Although research on EV-associated RNA has predominantly focused on microRNAs, the transcriptome of EV consists of multiple classes of small non-coding RNAs with potential gene-regulatory functions. It is not known whether environmental cues imposed on cells induce specific changes in a broad range of EV-associated RNA classes. Here, we investigated whether immune-activating or -suppressing stimuli imposed on primary dendritic cells affected the release of various small non-coding RNAs via EV. The small RNA transcriptomes of highly pure EV populations free from ribonucleoprotein particles were analyzed by RNA sequencing and RT-qPCR. Immune stimulus-specific changes were found in the miRNA, snoRNA, and Y-RNA content of EV from dendritic cells, whereas tRNA and snRNA levels were much less affected. Only part of the changes in EV-RNA content reflected changes in cellular RNA, which urges caution in interpreting EV as snapshots of cells. By comprehensive analysis of RNA obtained from highly purified EV, we demonstrate that multiple RNA classes contribute to genetic messages conveyed via EV. The identification of multiple RNA classes that display cell stimulation-dependent association with EV is the prelude to unraveling the function and biomarker potential of these EV-RNAs. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: MHC2/ CD63/ CD9/ Galectin-3
non-EV: beta-actin
Proteomics
no
Show all info
Study aim
Biomarker, Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
primary bone marrow dendritic cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
65
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Sample volume (mL)
0.15
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 40 Ti
Speed (g)
192000
Duration (min)
900-1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
65
Pelleting: rotor type
SW 40 Ti
Pelleting: speed (g)
192000
Pelleting: adjusted k-factor
144.0
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, MHC2, Galectin-3
Not detected contaminants
beta-actin
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100 - 200
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
see van der Vlist et al. 2012 Nature Protocols;and Nolte-'t Hoen 2012 Nanomedicine
Calibration bead size
0.1,0.2
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170013 1/1 Mus musculus endothelial progenitor cells DG
(d)(U)C
Degosserie, Jonathan 2018 100%

Study summary

Full title
All authors
Degosserie J, Heymans C, Spourquet C, Halbout M, D'Auria L, Van Der Smissen P, Vertommen D, Courtoy PJ, Tyteca D, Pierreux CE.
Journal
J Extracell Vesicles
Abstract
Organogenesis is a complex and dynamic process requiring reciprocal communication between different (show more...)Organogenesis is a complex and dynamic process requiring reciprocal communication between different cell types. In the thyroid, thyrocyte progenitors secrete the angiocrine factor, VEGFA, to recruit endothelial cells. In return, endothelial cells promote thyrocyte organisation into spherical follicular structures, which are responsible for thyroid hormone synthesis and storage. Medium conditioned by endothelial progenitor cells (EPCs) can promote follicle formation and lumen expansion (i.e. folliculogenesis) in an ex vivo culture system of thyroid lobes. Here, we postulated that endothelial cells instruct thyrocyte progenitors by producing extracellular vesicles (EVs). We found that medium conditioned by EPCs contain EVs with exosomal characteristics and that these vesicles can be incorporated into thyrocyte progenitors. By mass spectrometry, laminin peptides were abundantly identified in the EV preparations, probably co-sedimenting with EVs. Laminin-α1 silencing in EPC abrogated the folliculogenic effect of EVs. However, density gradient separation of EVs from laminins revealed that both EV-rich and laminin-rich fractions exhibited folliculogenic activity. In conclusion, we suggest that endothelial cells can produce EVs favouring thyrocyte organisation into follicles and lumen expansion, a mechanism promoted by laminin-α1. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
84.53 (pelleting) / 84.53 (washing)
Protein markers
EV: Flotillin-1/ CD63/ CD9
non-EV: Calnexin
Proteomics
yes
Show all info
Study aim
Function, Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
endothelial progenitor cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 80 Ti
Pelleting: speed (g)
150000
Pelleting: adjusted k-factor
84.53
Wash: time (min)
90
Wash: Rotor Type
Type 80 Ti
Wash: speed (g)
150000
Wash: adjusted k-factor
84.53
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
30%
Sample volume (mL)
1.3
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 55 Ti
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
0.625
Fraction processing
Centrifugation
Pelleting: volume per fraction
8
Pelleting: duration (min)
90
Pelleting: rotor type
Type 80 Ti
Pelleting: speed (g)
150000
Pelleting: adjusted k-factor
84.53
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-1
Not detected contaminants
Calnexin
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
100-150
EV concentration
Yes
Particle yield
3.60E+08 particles/million cells
EM
EM-type
Scanning-EM
Image type
Close-up, Wide-field
EV180010 2/2 Homo sapiens Serum DG
(d)(U)C
Busatto, Sara 2018 88%

Study summary

Full title
All authors
Sara Busatto, Arianna Giacomini, Costanza Montis, Roberto Ronca, Paolo Bergese
Journal
Anal Chem
Abstract
Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capi (show more...)Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth. (hide)
EV-METRIC
88% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Adj. k-factor
89.2 (pelleting)
Protein markers
EV: Alix/ CD81/ ANXA11/ CD63
non-EV: GM130/ APOA1
Proteomics
no
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
89.20
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0.15
Highest density fraction
0.6
Sample volume (mL)
0.8
Orientation
Bottom-up (sample migrates upwards)
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
0.4
Fraction processing
Centrifugation
Pelleting: volume per fraction
0.4
Pelleting: duration (min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, CD81, ANXA11
Not detected contaminants
GM130, APOA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
70
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
30-150
Other particle analysis name(2)
Colorimetric nanoplasmonic assay (CONAN)
EV-concentration
Yes
Particle yield
5.30E+12
EV180059 6/6 Gut microbiota Stool DG
SEC
Tulkens J 2018 87%

Study summary

Full title
All authors
Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A.
Journal
Gut
Abstract
(show more...) (hide)
EV-METRIC
87% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Stool
Sample origin
Control condition
Focus vesicles
gut bacteria-derived EV
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
SEC
Protein markers
EV: Proteomics/ Western blot/ LPS/ OmpA/ Limulus Amebocyte Lysate Assay
non-EV: Alix/ flotilin-1/ CD9
Proteomics
yes
EV density (g/ml)
1.141-1.186
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Gut microbiota
Sample Type
Stool
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10
Highest density fraction
50
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.667
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
OmpA
Not detected contaminants
Alix/ flotilin-1/ CD9
Proteomics database
No
Detected EV-associated proteins
LPS
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-300
EV concentration
Yes
Particle yield
particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Wide-field
EV170041 1/1 Homo sapiens 'SW480 Dukes' type B, colorectal adenocarcinoma;SW620, colon; derived from metastatic site: lymph node DG Yingkuan Shao 2018 87%

Study summary

Full title
All authors
Yingkuan Shao, Ting Chen, Xi Zheng, Sheng Yang, Kailun Xu, Xuewen Chen, Fei Xu, Lantian Wang, Yanwei Shen, Tingyang Wang, Mengwen Zhang, Wangxiong Hu, Chenyang Ye, XiaoFang Yu, Jimin Shao, Shu Zheng
Journal
Carcinogenesis
Abstract
Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are asso (show more...)Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs' induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21-TLR7-IL-6 axis. Thus, sEVs-miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis. (hide)
EV-METRIC
87% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
Protein markers
EV: Alix/ HSP70/ CD63/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Function, Biomarker, Mechanism of uptake/transfer, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
'SW480 Dukes' type B, colorectal adenocarcinoma / SW620, colon / derived from metastatic site: lymph node
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability (%)
NA
Separation Method
Density gradient
Only used for validation of main results
Yes
Density medium
69.83
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
0.25M
Highest density fraction
1.3M
Sample volume (mL)
1
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
150
Fraction volume (mL)
2
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
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
69.83
Pelleting-wash: speed (g)
MLA-130
Pelleting-wash: adjusted k-factor
69.83
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
2-5
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD9, CD63, HSP70
Not detected contaminants
Calreticulin
Characterization: RNA analysis
Database
Yes
Proteinase treatment
Yes
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
10
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
10
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-160
EV concentration
Yes
Particle yield
1.0-2.0E11
EM
EM-type
Transmission-EM
Image type
Wide-field
Report size (nm)
30-160
EV concentration
Yes
EV180083 1/2 Mus musculus 5TGM1 DG
(d)(U)C
ExoQuick
Faict S 2018 78%

Study summary

Full title
All authors
Faict S, Muller J, De Veirman K, De Bruyne E, Maes K, Vrancken L, Heusschen R, De Raeve H, Schots R, Vanderkerken K, Caers J, Menu E.
Journal
blood cancer j
Abstract
Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment w (show more...)Progression of multiple myeloma (MM) is largely dependent on the bone marrow (BM) microenvironment wherein communication through different factors including extracellular vesicles takes place. This cross-talk not only leads to drug resistance but also to the development of osteolysis. Targeting vesicle secretion could therefore simultaneously ameliorate drug response and bone disease. In this paper, we examined the effects of MM exosomes on different aspects of osteolysis using the 5TGM1 murine model. We found that 5TGM1 sEVs, or 'exosomes', not only enhanced osteoclast activity, they also blocked osteoblast differentiation and functionality in vitro. Mechanistically, we could demonstrate that transfer of DKK-1 led to a reduction in Runx2, Osterix, and Collagen 1A1 in osteoblasts. In vivo, we uncovered that 5TGM1 exosomes could induce osteolysis in a similar pattern as the MM cells themselves. Blocking exosome secretion using the sphingomyelinase inhibitor GW4869 not only increased cortical bone volume, but also it sensitized the myeloma cells to bortezomib, leading to a strong anti-tumor response when GW4869 and bortezomib were combined. Altogether, our results indicate an important role for exosomes in the BM microenvironment and suggest a novel therapeutic target for anti-myeloma therapy. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
ExoQuick
Protein markers
EV: / TSG101/ CD63/ Syntenin/ CD81
non-EV: / Calreticulin
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
5TGM1
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
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 41 Ti
Speed (g)
100000
Duration (min)
10800
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
10
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ TSG101/ Syntenin/ CD81
Not detected EV-associated proteins
Detected contaminants
Not detected contaminants
Calreticulin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
114
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
100
EV190002 1/6 Homo sapiens MIA PaCa-2 DG
(d)(U)C
Filtration
Sachiko Matsumura 2018 78%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin
non-EV: Histon H2B
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MIA PaCa-2
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
< 1.06 g/ml
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ MFGE8/ beta-actin/ HSP70/ CD81
Not detected EV-associated proteins
TSG101/ Alix
Not detected contaminants
Histon H2B
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
136
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
height: 39.1, diameter: 135.9
EV190002 2/6 Homo sapiens MIA PaCa-2 DG
(d)(U)C
Filtration
Sachiko Matsumura 2018 78%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin
non-EV: Histon H2B
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MIA PaCa-2
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
ca. 1.1 g/ml
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD63/ MFGE8/ beta-actin/ TSG101/ HSP70/ CD81
Not detected contaminants
Histon H2B
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
124
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
height: 30.1, diameter: 96.4
EV190002 3/6 Homo sapiens MIA PaCa-2 DG
(d)(U)C
Filtration
Sachiko Matsumura 2018 78%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ Src/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin/ CD9
non-EV: Argonaute2/ Histon H2B
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MIA PaCa-2
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
< 1.06 g/ml
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ HSP70/ MFGE8/ Src/ beta-actin/ CD81
Not detected EV-associated proteins
TSG101/ Alix
Not detected contaminants
Histon H2B/ Argonaute2
Detected EV-associated proteins
CD63
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
148
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
height: 50.5, length: 190.0
EV190002 4/6 Homo sapiens MIA PaCa-2 DG
(d)(U)C
Filtration
Sachiko Matsumura 2018 78%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
78% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ Src/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin/ CD9
non-EV: Argonaute2/ Histon H2B
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MIA PaCa-2
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
ca. 1.1 g/ml
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ MFGE8/ Src/ beta-actin/ CD9/ CD63/ TSG101/ HSP70/ CD81
Not detected contaminants
Histon H2B/ Argonaute2
Detected EV-associated proteins
CD9/ CD63
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
127
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
height 32.1, length: 120.0
EV180026 5/7 Homo sapiens Serum (d)(U)C Wenzhe Li 2018 77%

Study summary

Full title
All authors
Wenzhe Li, Bin Shao, Changliang Liu, Huayi Wang, Wangshu Zheng, Weiyao Kong, Xiaoran Liu, Guobin Xu, Chen Wang, Huiping Li, Ling Zhu, Yanlian Yang
Journal
Small methods
Abstract
Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dyn (show more...)Blood‐based detection and molecular phenotyping are highly desired for the early diagnosis and dynamic monitoring of cancer. Extracellular vesicles (EVs) carry molecular information from the cells of origin and are biomarkers of cancer. However, the detection and molecular analysis of EVs has been challenging due to their nanoscaled size. Here, an assessment of the detection and molecular phenotyping of serum EVs based on microbead‐assisted flow cytometry is established. The clinical utility of this method is validated in the diagnosis and human epidermal growth factor receptor 2 (HER2) phenotyping of breast cancer. Good correlation between the status of epithelial cell adhesion molecule (EpCAM) and HER2 expression in EVs and in the cells of origin is found. Both EpCAM+ and HER2+ EVs are demonstrated to be effective diagnostic markers of breast cancer with high sensitivity and specificity. EV‐based HER2 phenotyping is consistent with tissue‐based HER2 phenotyping by immunohistochemistry and can be used as a surrogate for the invasive tissue assessments. The microbead‐assisted flow cytometry assessment of EVs enables rapid and noninvasive detection and molecular phenotyping of cancer and would help to personalized treatment and cancer survival. (hide)
EV-METRIC
77% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
120.7 (pelleting) / 120.7 (washing)
Protein markers
EV: CD81/ Flotillin-1/ CD63/ HER2/ EpCAM
non-EV: Calnexin/ Albumin
Proteomics
no
Show all info
Study aim
Function, Biomarker, New methodological development
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
1200
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
130000
Pelleting: adjusted k-factor
120.7
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
130000
Wash: adjusted k-factor
120.7
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
30-120
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, Flotillin-1
Not detected contaminants
Albumin, Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Size range/distribution
Reported size (nm)
110.2 ± 12.95 nm
NTA
Report type
Size range/distribution
Reported size (nm)
124.4 ± 53.3 nm
EV concentration
Yes
Particle yield
3290000000000
EM
EM-type
Transmission-EM/ Immune-EM
EM protein
EpCAM,HER2
Image type
Close-up, Wide-field
EV concentration
Yes
EV170020 1/2 Trypanosoma cruzi Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA DG
(d)(U)C
Filtration
Caeiro, Lucas 2018 77%

Study summary

Full title
All authors
Caeiro LD, Alba-Soto CD, Rizzi M, Solana ME, Rodriguez G, Chidichimo AM, Rodriguez ME, Sánchez DO, Levy GV, Tekiel V.
Journal
PLoS Negl Trop Dis
Abstract
TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage (show more...)TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes. (hide)
EV-METRIC
77% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: TcTASV-C/ HSP70/ HSP70,TcTASV-C
non-EV: TcSR-62
Proteomics
yes
Show all info
Study aim
Biomarker, Identification of content (omics approaches)
Sample
Species
Trypanosoma cruzi
Sample Type
Cell culture supernatant
EV-producing cells
Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA
EV-harvesting Medium
Serum free medium
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
2
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
0.1
Western Blot
Antibody details provided?
No
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70,TcTASV-C
Not detected contaminants
TcSR-62
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-150
EV170020 2/2 Trypanosoma cruzi Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA DG
(d)(U)C
Filtration
Caeiro, Lucas 2018 77%

Study summary

Full title
All authors
Caeiro LD, Alba-Soto CD, Rizzi M, Solana ME, Rodriguez G, Chidichimo AM, Rodriguez ME, Sánchez DO, Levy GV, Tekiel V.
Journal
PLoS Negl Trop Dis
Abstract
TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage (show more...)TcTASV-C is a protein family of about 15 members that is expressed only in the trypomastigote stage of Trypanosoma cruzi. We have previously shown that TcTASV-C is located at the parasite surface and secreted to the medium. Here we report that the expression of different TcTASV-C genes occurs simultaneously at the trypomastigote stage and while some secreted and parasite-associated products are found in both fractions, others are different. Secreted TcTASV-C are mainly shedded through trypomastigote extracellular vesicles, of which they are an abundant constituent, despite its scarce expression on culture-derived trypomastigotes. In contrast, TcTASV-C is highly expressed in bloodstream trypomastigotes; its upregulation in bloodstream parasites was observed in different T. cruzi strains and was specific for TcTASV-C, suggesting that some host-molecules trigger TcTASV-C expression. TcTASV-C is also strongly secreted by bloodstream parasites. A DNA prime-protein boost immunization scheme with TcTASV-C was only partially effective to control the infection in mice challenged with a highly virulent T. cruzi strain. Vaccination triggered a strong humoral response that delayed the appearance of bloodstream trypomastigotes at the early phase of the infection. Linear epitopes recognized by vaccinated mice were mapped within the TcTASV-C family motif, suggesting that blockade of secreted TcTASV-C impacts on the settlement of infection. Furthermore, although experimental and naturally T. cruzi-infected hosts did not react with antigens from extracellular vesicles, vaccinated and challenged mice recognized not only TcTASV-C but also other vesicle-antigens. We hypothesize that TcTASV-C is involved in the establishment of the initial T. cruzi infection in the mammalian host. Altogether, these results point towards TcTASV-C as a novel secreted virulence factor of T. cruzi trypomastigotes. (hide)
EV-METRIC
77% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: TcTASV-C/ HSP70/ HSP70,TcTASV-C
non-EV: TcSR-62
Proteomics
yes
Show all info
Study aim
Biomarker, Identification of content (omics approaches)
Sample
Species
Trypanosoma cruzi
Sample Type
Cell culture supernatant
EV-producing cells
Trypomastigote CL-Brener, Trypomastigote Sylvio, Trypomastigote Y, Trypomastigote RA
EV-harvesting Medium
Serum free medium
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
1080
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
156.9
Wash: time (min)
1080
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
100000
Wash: adjusted k-factor
156.9
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
2
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
0.15
Western Blot
Antibody details provided?
No
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70,TcTASV-C
Not detected contaminants
TcSR-62
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
50-150
EV180059 1/6 Gram-negative bacteria Escherichia coli Nissle 1917 DG
SEC
Tulkens J 2018 75%

Study summary

Full title
All authors
Tulkens J, Vergauwen G, Van Deun J, Geeurickx E, Dhondt B, Lippens L, De Scheerder MA, Miinalainen I, Rappu P, De Geest BG, Vandecasteele K, Laukens D, Vandekerckhove L, Denys H, Vandesompele J, De Wever O, Hendrix A.
Journal
Gut
Abstract
(show more...) (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Escherichia coli
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
SEC
Protein markers
EV: Limulus Amebocyte Lysate assay/ LPS/ OmpA/ Western blot
non-EV:
Proteomics
no
EV density (g/ml)
1.141-1.186
Show all info
Study aim
Function/Biomarker/Identification of content (omics approaches)
Sample
Species
Gram-negative bacteria
Sample Type
Cell culture supernatant
EV-producing cells
Escherichia coli Nissle 1917
EV-harvesting Medium
Serum free medium
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10
Highest density fraction
50
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.667
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
2
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
OmpA/ LPS
Proteomics database
No
Detected EV-associated proteins
LPS
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-300
EV concentration
Yes
EM
EM-type
Immuno-EM
EM protein
LPS
Image type
Close-up, Wide-field
EV180053 1/1 Homo sapiens MKN74, MKN45 (d)(U)C
Filtration
Rocha, Sara 2018 75%

Study summary

Full title
All authors
Sara Rocha, Joana Carvalho, Patrícia Oliveira, Maren Voglstaetter, Domitille Schvartz, Andreas R. Thomsen, Nadia Walter, Richa Khanduri, Jean‐Charles Sanchez, Andreas Keller, Carla Oliveira, Irina Nazarenko
Journal
Advanced Science
Abstract
The success of malignant tumors is conditioned by the intercellular communication between tumor cell (show more...)The success of malignant tumors is conditioned by the intercellular communication between tumor cells and their microenvironment, with extracellular vesicles (EVs) acting as main mediators. While the value of 3D conditions to study tumor cells is well established, the impact of cellular architecture on EV content and function is not investigated yet. Here, a recently developed 3D cell culture microwell array is adapted for EV production and a comprehensive comparative analysis of biochemical features, RNA and proteomic profiles of EVs secreted by 2D vs 3D cultures of gastric cancer cells, is performed. 3D cultures are significantly more efficient in producing EVs than 2D cultures. Global upregulation of microRNAs and downregulation of proteins in 3D are observed, indicating their dynamic coregulation in response to cellular architecture, with the ADP‐ribosylation factor 6 signaling pathway significantly downregulated in 3D EVs. The data strengthen the biological relevance of cellular architecture for production and cargo of EVs. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: integrin-alpha6/ catenin-beta1/ integrin-alpha2/ integrin-alpha3/ tubulin/ Flotillin-1/ E-cadherin/ GAPDH/ CD81/ integrin-beta1/ catenin-delta1/ CD9
non-EV: CytochromeC
Proteomics
yes
Show all info
Study aim
New methodological development, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MKN74, MKN45
EV-harvesting Medium
Serum free medium
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
0.2
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
integrin-alpha2, integrin-alpha3, integrin-alpha6, integrin-beta1, E-cadherin, catenin-beta1, catenin-delta1, GAPDH, tubulin
Proteomics database
Yes
Characterization: RNA analysis
Database
Yes
Proteinase treatment
Yes
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
5
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
1
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
110
EV concentration
Yes
Particle yield
1.50E+09 particles/million cells
Particle analysis: flow cytometry
Flow cytometer type
Amnis ImageStream
Hardware adjustment
Calibration bead size
200
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
110
EV180033 1/3 Homo sapiens Lipoaspirate (d)(U)C
Tangential flow filtration
Busatto S 2018 75%

Study summary

Full title
All authors
Busatto S, Vilanilam G, Ticer T, Lin WL, Dickson DW, Shapiro S, Bergese P, Wolfram J1.
Journal
Cells
Abstract
Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible (show more...)Concentration of extracellular vesicles (EVs) from biological fluids in a scalable and reproducible manner represents a major challenge. This study reports the use of tangential flow filtration (TFF) for the highly efficient isolation of EVs from large volumes of samples. When compared to ultracentrifugation (UC), which is the most widely used method to concentrate EVs, TFF is a more efficient, scalable, and gentler method. Comparative assessment of TFF and UC of conditioned cell culture media revealed that the former concentrates EVs of comparable physicochemical characteristics, but with higher yield, less single macromolecules and aggregates (<15 nm in size), and improved batch-to-batch consistency in half the processing time (1 h). The TFF protocol was then successfully implemented on fluids derived from patient lipoaspirate. EVs from adipose tissue are of high clinical relevance, as they are expected to mirror the regenerative properties of the parent cells. (hide)
EV-METRIC
75% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Lipoaspirate
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Tangential flow filtration
Protein markers
EV: CD81/ CD63/ CD9
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Lipoaspirate
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Other
Name other separation method
Tangential flow filtration
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, CD81
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
170-185
EV concentration
Yes
Particle yield
20000000000
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV180008 1/4 Homo sapiens HCCLM3 DG
(d)(U)C
Hao, Liu 2018 75%

Study summary

Full title
All authors
Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang
Journal
Oncogene
Abstract
Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: CD81/ TSG101/ CD63
non-EV: Grp94/ Argonaute-2
Proteomics
no
Show all info
Study aim
Function, Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HCCLM3
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Density medium
255.8
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Pelleting-wash: volume per pellet (mL)
12
Pelleting-wash: duration (min)
180
Pelleting-wash: rotor type
255.8
Pelleting-wash: speed (g)
SW 41 Ti
Pelleting-wash: adjusted k-factor
255.8
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
0.38
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, TSG101
Not detected contaminants
Grp94
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180008 4/4 Homo sapiens HuH7 DG
(d)(U)C
Hao, Liu 2018 75%

Study summary

Full title
All authors
Hao Liu, Wei Chen, Xiao Zhi, En-Jiang Chen, Tao Wei, Jian Zhang, Jian Shen, Li-Qiang Hu, Bin Zhao, Xin-Hua Feng, Xue-Li Bai, Ting-Bo Liang
Journal
Oncogene
Abstract
Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The proc (show more...)Tumor self-seeding occurs when circulating malignant cells reinfiltrate the original tumor. The process may breed more aggressive tumor cells, which may contribute to cancer progression. In this study, we observed tumor self-seeding in mouse xenograft models of hepatocellular carcinoma (HCC) for the first time. We confirmed that circulating tumor cell uptake of tumor-derived exosomes, which are increasingly recognized as key instigators of cancer progression by facilitating cell-cell communication, promoted tumor self-seeding by enhancing the invasive and migration capability of recipient HCC cells. Horizontal transfer of exosomal microRNA-25-5p to anoikis-resistant HCC cells significantly enhanced their migratory and invasive abilities, whereas inhibiting microRNA-25-5p alleviated these effects. Our experiments delineate an exosome-based novel pathway employed by functional microRNA from the original tumor cells that can influence the biological fate of circulating tumor cells. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: CD81/ TSG101/ CD63
non-EV: Grp94/ Argonaute-2
Proteomics
no
Show all info
Study aim
Function, Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HuH7
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Density medium
255.8
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Pelleting-wash: volume per pellet (mL)
12
Pelleting-wash: duration (min)
180
Pelleting-wash: rotor type
255.8
Pelleting-wash: speed (g)
SW 41 Ti
Pelleting-wash: adjusted k-factor
255.8
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
0.12
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, TSG101
Not detected contaminants
Grp94
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Transmission-EM
Image type
Wide-field
YJ7428AP 1/2 Homo sapiens Unconditioned serum-containing medium (d)(U)C
Filtration
qEV
UF
Sjöqvist S 2018 75%

Study summary

Full title
All authors
Sjöqvist S, Ishikawa T, Shimura D, Kasai Y, Imafuku A, Bou-Ghannam S, Iwata T, Kanai N
Journal
J Extracell Vesicles
Abstract
The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound (show more...)The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer. (hide)
EV-METRIC
75% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Unconditioned serum-containing medium
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
qEV
UF
Protein markers
EV: Flotillin-1/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Unconditioned serum-containing medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10, 100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
1.905
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1
Not detected contaminants
GRP94
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
119.2±4.5
EV concentration
Yes
Particle yield
2.035E09 ± 3.735E08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
YJ7428AP 2/2 Homo sapiens primary mucosal epithelial cells (d)(U)C
Filtration
qEV
UF
Sjöqvist S 2018 75%

Study summary

Full title
All authors
Sjöqvist S, Ishikawa T, Shimura D, Kasai Y, Imafuku A, Bou-Ghannam S, Iwata T, Kanai N
Journal
J Extracell Vesicles
Abstract
The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound (show more...)The oral mucosa exhibits unique regenerative properties, sometimes referred to as foetal-like wound healing. Researchers from our institute have used sheets of oral mucosa epithelial cells (OMECs) for regenerative medicine applications including cornea replacement and oesophageal epithelial regeneration for stricture prevention. Here, we have isolated exosomes from clinical-grade production of OMEC sheets from healthy human donors (n = 8), aiming to evaluate the clinical potential of the exosomes to stimulate epithelial regeneration and to improve understanding of the mode-of-action of the cells. Exosomes were isolated from conditioned (cExo) and non-conditioned (ncExo) media. Characterization was performed using Western blot for common exosomal-markers: CD9 and flotillin were positive while annexin V, EpCam and contaminating marker GRP94 were negative. Nanoparticle tracking analysis revealed a diameter of ~120 nm and transmission electron microscopy showed a corresponding size and spherical appearance. Human skin fibroblasts exposed to exosomes showed dose-dependent reduction of proliferation and a considerable increase of growth factor gene expression (HGF, VEGFA, FGF2 and CTGF). The results were similar for both groups, but with a trend towards a larger effect from cExo. To study adhesion, fluorescently labelled exosomes were topically applied to pig oesophageal wound-beds ex vivo and subsequently washed. Positive signal could be detected after as little as 1 min of adhesion, but increased adhesion time produced a stronger signal. Next, labelled exosomes were added to full-thickness skin wounds in rats and signal was detected up to 5 days after application. cExo significantly reduced the wound size at days 6 and 17. In conclusion, exosomes from OMEC sheets showed pro-regenerative effects on skin wound healing. This is the first time that the healing capacity of the oral mucosa is studied from an exosome perspective. These findings might lead to a combinational therapy of cell sheets and exosomes for future patients with early oesophageal cancer. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
qEV
UF
Protein markers
EV: Flotillin-1/ CD9
non-EV: GRP94
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary mucosal epithelial cells
EV-harvesting Medium
Serum-containing medium
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Filtration steps
0.22µm or 0.2µm
Ultra filtration
Cut-off size (kDa)
10, 100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Protein Yield (µg)
0.97
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, Flotillin-1
Not detected contaminants
GRP94
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
124.8±4.1
EV concentration
Yes
Particle yield
1.027E09 ± 9.279E08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV170014 1/4 Homo sapiens HEK293 (d)(U)C
Filtration
SEC
Tangential flow filtration (50 nm pore size)
Dionysios C Watson 2018 75%

Study summary

Full title
All authors
Dionysios C Watson, Bryant C Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C Jones, Barbara K Felber, Xiaoyuan Chen, George N Pavlakis
Journal
J Extracell Vesicles
Abstract
The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the e (show more...)The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
human hetIL-15 stably transfected,human hetIL-15/lactadherin stably transfected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
SEC
Tangential flow filtration (50 nm pore size)
Protein markers
EV: IL-15/ hetIL-15/lactadherin/ CD63
non-EV: calnexin/ ferritin
Proteomics
yes
Show all info
Study aim
New methodological development, Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HEK293
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting performed
No
Size-exclusion chromatography
Total column volume (mL)
120
Sample volume/column (mL)
5
Resin type
Superdex 200
Other
Name other separation method
Tangential flow filtration (50 nm pore size)
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected contaminants
calnexin
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
No
Selected surface protein(s)
CD63
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-200
EV concentration
Yes
Particle yield
480000000000
EM
EM-type
Immune-EM
EM protein
IL-15
Image type
Close-up, Wide-field
EV220208 1/2 Homo sapiens HUVEC Filtration
dUC
Hosseinkhani B 2018 67%

Study summary

Full title
All authors
Hosseinkhani B, Kuypers S, van den Akker NMS, Molin DGM, Michiels L
Journal
Front Immunol
Abstract
Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial c (show more...)Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) . Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
dUC
Protein markers
EV: CD63/ ICAM-1/ IL-6, IL-8, ICAM-1, CCL-2/ CD9/ IL-8/ TIMP2/ ICAM-1/ IL6-R/ CXCL10/ CCL-2/ PDGF-BB
non-EV: GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Other
Name other separation method
dUC
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ ICAM-1/ ?-Actin
Not detected contaminants
GM130
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
IL-6, IL-8, ICAM-1, CCL-2
Other 1
ELISA
Detected EV-associated proteins
IL-8/ TIMP2/ ICAM-1/ IL6-R/ CXCL10/ CCL-2/ PDGF-BB
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV210198 3/7 Homo sapiens A172 (d)(U)C
Filtration
Hu, Guoku 2018 67%

Study summary

Full title
All authors
Guoku Hu, Ke Liao, Fang Niu, Lu Yang, Blake W Dallon, Shannon Callen, Changhai Tian, Jiang Shu, Juan Cui, Zhiqiang Sun, Yuri L Lyubchenko, Minhan Ka, Xian-Ming Chen, Shilpa Buch
Journal
Mol Ther Nucleic Acids
Abstract
Impairment of microglial functions, such as phagocytosis and/or dysregulation of immune responses, h (show more...)Impairment of microglial functions, such as phagocytosis and/or dysregulation of immune responses, has been implicated as an underlying factor involved in the pathogenesis of various neurodegenerative disorders. Our previous studies have demonstrated that long intergenic noncoding RNA (lincRNA)-Cox2 expression is influenced by nuclear factor κB (NF-κB) signaling and serves as a coactivator of transcriptional factors to regulate the expression of a vast array of immune-related genes in microglia. Extracellular vesicles (EVs) have been recognized as primary facilitators of cell-to-cell communication and cellular regulation. Herein, we show that EVs derived from astrocytes exposed to morphine can be taken up by microglial endosomes, leading, in turn, to activation of Toll-like receptor 7 (TLR7) with a subsequent upregulation of lincRNA-Cox2 expression, ultimately resulting in impaired microglial phagocytosis. This was further validated in vivo, wherein inhibition of microglial phagocytic activity was also observed in brain slices isolated from morphine-administrated mice compared with control mice. Additionally, we also showed that intranasal delivery of EVs containing lincRNA-Cox2 siRNA (small interfering RNA) was able to restore microglial phagocytic activity in mice administered morphine. These findings have ramifications for the development of EV-loaded RNA-based therapeutics for the treatment of various disorders involving functional impairment of microglia. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: Alix/ TSG101/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
A172
EV-harvesting Medium
Not specified
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Alix/ CD63/ TSG101
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
80
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200136 2/7 Homo sapiens Blood plasma DG
(d)(U)C
Filtration
Miranda, Jezid 2018 67%

Study summary

Full title
All authors
Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon
Journal
Placenta
Abstract
Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Maternal; Normal pregnancy
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ Flotillin1/ PLAP/ CD63
non-EV: Grp94
Proteomics
no
EV density (g/ml)
1.12-1.188g/ml
Show all info
Study aim
Function/Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
T-8100
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
120
Wash: Rotor Type
T-8100
Wash: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Sample volume (mL)
0.5mL
Orientation
Bottom-up
Rotor type
T-8100
Speed (g)
100000
Duration (min)
1200
Fraction processing
Centrifugation
Pelleting: volume per fraction
0.05
Pelleting: duration (min)
120
Pelleting: rotor type
T-8100
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ TSG101
Not detected contaminants
Grp94
ELISA
Antibody details provided?
No
Detected EV-associated proteins
PLAP
Fluorescent NTA
Relevant measurements variables specified?
NA
Antibody details provided?
No
Detected EV-associated proteins
PLAP/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
87+/-23
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200136 5/7 Homo sapiens Blood plasma DG
(d)(U)C
Filtration
Miranda, Jezid 2018 67%

Study summary

Full title
All authors
Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon
Journal
Placenta
Abstract
Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Fetal; Normal pregnancy
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ Flotillin1/ PLAP/ CD63
non-EV: Grp94
Proteomics
no
EV density (g/ml)
1.12-1.188g/ml
Show all info
Study aim
Function/Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
T-8100
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
120
Wash: Rotor Type
T-8100
Wash: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Sample volume (mL)
0.5mL
Orientation
Bottom-up
Rotor type
T-8100
Speed (g)
100000
Duration (min)
1200
Fraction processing
Centrifugation
Pelleting: volume per fraction
0.05
Pelleting: duration (min)
120
Pelleting: rotor type
T-8100
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ TSG101
Not detected contaminants
Grp94
ELISA
Antibody details provided?
No
Detected EV-associated proteins
PLAP
Fluorescent NTA
Relevant measurements variables specified?
NA
Antibody details provided?
No
Detected EV-associated proteins
PLAP/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
90 ± 17 nm
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190002 5/6 Homo sapiens HT-29 DG
(d)(U)C
Filtration
Sachiko Matsumura 2018 67%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: CD81/ EpCAM/ CD63/ CD9
non-EV:
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HT-29
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
< 1.06 g/ml
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
EpCAM/ CD9
Not detected EV-associated proteins
CD81/ CD63
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
162
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
EV190002 6/6 Homo sapiens HT-29 DG
(d)(U)C
Filtration
Sachiko Matsumura 2018 67%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: CD81/ EpCAM/ CD63/ CD9
non-EV:
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HT-29
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
ca. 1.1 g/ml
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ EpCAM/ CD81
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
142
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
EV180030 3/7 Homo sapiens PM1 DG
(d)(U)C
Zhaohao Liao 2018 67%

Study summary

Full title
All authors
Zhaohao Liao, Lorena Martin Jaular ORCID Icon, Estelle Soueidi, Mabel Jouve, Dillon C. Muth, Tine H. Schøyen, Tessa Seale, Norman J. Haughey, Matias Ostrowski, Clotilde Théry ORCID Icon & Kenneth W. Witwer
Journal
J Extracell Vesicles
Abstract
Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was deve (show more...)Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum “extra-depletion” protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
HIV-BaL infected
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: / CD81/ CD63/ CD9
non-EV: AChE
Proteomics
no
EV density (g/ml)
Not specified
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
PM1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
50
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
11
Lowest density fraction
6%
Highest density fraction
18%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
1
Orientation
Top-down
Rotor type
TH-641
Speed (g)
210000
Duration (min)
120
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
35
Pelleting: duration (min)
40
Pelleting: rotor type
AH-629 (36 ml)
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ CD81
Other 1
Acetylcholinesterase assay
Detected EV-associated proteins
Not detected contaminants
AChE
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV180022 1/4 Homo sapiens pluripotent stem cells (d)(U)C Kaur S 2018 67%

Study summary

Full title
All authors
Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B
Journal
J Cell Sci
Abstract
Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
208.3 (pelleting) / 208.3 (washing)
Protein markers
EV: TSG101/ HSP70/ CD63/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
pluripotent stem cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
121896
Pelleting: adjusted k-factor
208.3
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
121896
Wash: adjusted k-factor
208.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, HSP70, TSG101, CD90
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
101-500
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180022 4/4 Homo sapiens pluripotent stem cells miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark) Kaur S 2018 67%

Study summary

Full title
All authors
Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B
Journal
J Cell Sci
Abstract
Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
pluripotent stem cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
NA
Separation Method
Commercial kit
miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark)
Other
Name other separation method
miRCURY Exosome Isolation Kit (Exiqon A/S, Vedbaek, Denmark)
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Characterization: RNA analysis
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
EV180051 2/2 Rattus norvegicus Blood plasma (d)(U)C Takov K 2018 66%

Study summary

Full title
All authors
Takov K, Yellon DM, Davidson SM
Journal
J Extracell Vesicles
Abstract
Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids (show more...)Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB+ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium. (hide)
EV-METRIC
66% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
small extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Adj. k-factor
115.3 (pelleting) / 115.3 (washing)
Protein markers
EV: CD81/ HSP70/ Endothelin-1/ CD9
non-EV: ApoB
Proteomics
no
Show all info
Study aim
Function, Technical analysis comparing/optimizing EV-related methods, Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
MLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
115.3
Wash: time (min)
70
Wash: Rotor Type
MLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
115.3
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
13.6-14.6
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70
ELISA
Antibody details provided?
Yes
Other 1
Protein array (ARY007, R&D Systems)
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
96.6
EV concentration
Yes
Particle yield
20000000000
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180022 3/4 Homo sapiens adipose tissue-derived mesenchymal stem cells (d)(U)C
Filtration
Kaur S 2018 66%

Study summary

Full title
All authors
Kaur S, Abu-Shahba AG, Paananen RO, Hongisto H, Hiidenmaa H, Skottman H, Seppänen-Kaijansinkko R, Mannerström B
Journal
J Cell Sci
Abstract
Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and (show more...)Extracellular vesicles (EVs) are reported to be involved in stem cell maintenance, self-renewal, and differentiation. Due to their bioactive cargoes influencing cell fate and function, interest in EVs in regenerative medicine has rapidly increased. EV-derived small non-coding RNA mimic the functions of the parent stem cells, regulating the maintenance and differentiation of stem cells, controlling the intercellular regulation of gene expression, and eventually affecting the cell fate. In this study, we used RNA sequencing to provide a comprehensive overview of the expression profiles of small non-coding transcripts carried by the EVs derived from human adipose tissue stromal/stem cells (AT-MSCs) and human pluripotent stem cells (hPSCs), both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSC). Both hPSCs and AT-MSCs were characterized and their EVs were extracted using standard protocols. Small non-coding RNA sequencing from EVs showed that hPSCs and AT-MSCs showed distinct profiles, unique for each stem cell source. Interestingly, in hPSCs, most abundant miRNAs were from specific miRNA families regulating pluripotency, reprogramming and differentiation (miR-17-92, mir-200, miR-302/367, miR-371/373, CM19 microRNA cluster). For the AT-MSCs, the highly expressed miRNAs were found to be regulating osteogenesis (let-7/98, miR-10/100, miR-125, miR-196, miR-199, miR-615-3p, mir-22-3p, mir-24-3p, mir-27a-3p, mir-193b-5p, mir-195-3p). Additionally, abundant small nuclear and nucleolar RNA were detected in hPSCs, whereas Y- and tRNA were found in AT-MSCs. Identification of EV-miRNA and non-coding RNA signatures released by these stem cells will provide clues towards understanding their role in intracellular communication, and well as their roles in maintaining the stem cell niche. (hide)
EV-METRIC
66% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Adj. k-factor
208.3 (pelleting) / 208.3 (washing)
Protein markers
EV: TSG101/ CD63/ CD90
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
adipose tissue-derived mesenchymal stem cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
NA
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
121896
Pelleting: adjusted k-factor
208.3
Wash: time (min)
120
Wash: Rotor Type
SW 28
Wash: speed (g)
121896
Wash: adjusted k-factor
208.3
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, TSG101, CD90
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
101-500
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV210279 1/2 Homo sapiens A431 (d)(U)C
DG
Libregts SFWM 2018 63%

Study summary

Full title
All authors
Libregts SFWM, Arkesteijn GJA, Németh A, Nolte-'t Hoen ENM, Wauben MHM
Journal
J Thromb Haemost
Abstract
Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. F (show more...)Essentials Extracellular vesicles (EVs) in biological fluids are promising biomarkers for disease. Fluorescence-based flow cytometric analysis is suitable to detect low abundant EV subsets. Particles of non-interest can induce false-positive light scatter and fluorescent signals. Interference of particles of non-interest can be monitored by analyzing serial dilutions. (hide)
EV-METRIC
63% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: EGFR/ CD9/ CD142
non-EV: None
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
A431
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW28 or SW32
Pelleting: speed (g)
100000
Density gradient
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.08M
Total gradient volume, incl. sample (mL)
11.6
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 40 Ti
Speed (g)
270000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Flow cytometry
Type of Flow cytometry
BD Influx/ Beckman Coulter CytoFLEX LX
Hardware adaptation to ~100nm EV's
BD Influx: Adapted small particle detector
Calibration bead size
0.1 and 0.2
Antibody details provided?
No
Detected EV-associated proteins
CD9/ EGFR/ CD142
Characterization: Lipid analysis
No
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
BD Influx/ Beckman Coulter Cytoflex LX
Hardware adjustment
BD Influx: adapted small particle detector
Calibration bead size
0.1 and 0.2
EV concentration
Yes
EV180017 1/5 Homo sapiens MDAMB231 DG
(d)(U)C
Filtration
UF
Beghein E 2018 63%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
63% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
UF
Protein markers
EV: TSG101/ CD63/ CD9/ MT1-MMP/ Flotillin-2
non-EV: Calnexin/ Golgin245/ PMP70/ CytochromeC
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
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
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63, Flotillin-2, TSG101, MT1-MMP
Not detected contaminants
PMP70, CytochromeC, Calnexin, Golgin245
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
131
EV concentration
Yes
Particle yield
4.95E+09 particles/million cells
EV180051 1/2 Rattus norvegicus Blood plasma (d)(U)C
qEV
UF
Takov K 2018 62%

Study summary

Full title
All authors
Takov K, Yellon DM, Davidson SM
Journal
J Extracell Vesicles
Abstract
Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids (show more...)Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB+ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium. (hide)
EV-METRIC
62% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
small extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
qEV
UF
Protein markers
EV: CD81/ HSP70/ Endothelin-1/ CD9
non-EV: ApoB
Proteomics
no
Show all info
Study aim
Function, Technical analysis comparing/optimizing EV-related methods, Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Pelleting performed
No
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
39-308
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70
ELISA
Antibody details provided?
Yes
Other 1
Protein array (ARY007, R&D Systems)
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
81.5
EV concentration
Yes
Particle yield
2000000000000
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180017 2/5 Homo sapiens MDAMB231 DG
(d)(U)C
Filtration
UF
Beghein E 2018 62%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
62% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Expressing fascin nanobody
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
UF
Protein markers
EV: CD63/ CD9
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
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
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
142
EV concentration
Yes
Particle yield
2.25E+10 particles/million cells
EV180017 5/5 Homo sapiens MDAMB231 DG
(d)(U)C
Filtration
UF
Beghein E 2018 62%

Study summary

Full title
All authors
Beghein E, Devriese D, Van Hoey E, Gettemans J
Journal
J Cell Sci
Abstract
Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasi (show more...)Cancer cell-derived extracellular vesicles (EVs) are increasingly being recognized as genuine invasive structures as they contribute to many aspects of invasion and metastasis. Unfortunately, the mechanisms underlying EV biogenesis or release are still poorly understood. Recent reports however indicate a role of the actin cytoskeleton in this process. In this study, we have exploited thoroughly characterized camelid nanobodies against actin binding proteins cortactin and fascin-1, a branched actin regulator and actin bundler, respectively, in order to assess their roles in EV biogenesis or release. Using this strategy, we demonstrate a role of the cortactin NTA and SH3 domains in EV release. Fascin-1 also regulates EV release, independently of its actin-bundling activity. We show a contribution of these protein domains in endosomal trafficking, a crucial step in EV biogenesis, and we confirm that EVs are preferentially released at invadopodia, the latter being actin-rich invasive cell protrusions in which cortactin and fascin-1 perform essential roles. Accordingly, EVs are enriched with invadopodial proteins such as the matrix metalloproteinase MT1-MMP and exert gelatinolytic activity. Based on our findings, we report that both cortactin and fascin-1 play key roles in EV release by regulating endosomal trafficking or invadopodia formation and function. (hide)
EV-METRIC
62% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Expressing cortactin nanobody (NTA domain)
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
UF
Protein markers
EV: CD63/ CD9
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/cargo sorting, Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDAMB231
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
0.5
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
11
Pelleting: duration (min)
180
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
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
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9, CD63
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
133
EV concentration
Yes
Particle yield
1.16E+09 particles/million cells
EV170025 1/1 Homo sapiens Urine qEV
UF
Oeyen E 2018 62%

Study summary

Full title
All authors
Oeyen E, Van Mol K, Baggerman G, Willems H, Boonen K, Rolfo C, Pauwels P, Jacobs A, Schildermans K, Cho WC, Mertens I.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolati (show more...)Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs. (hide)
EV-METRIC
62% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
qEV
UF
Protein markers
EV: Flotillin-1
non-EV: None
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches), Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Separation Method
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
0.81
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Flotillin-1
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
108
EV concentration
Yes
Particle yield
1.80E+09 particles/ml start sample
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
40-100
Other particle analysis name(1)
Asymmetrical flow field-flow fractionation
Report type
Size range/distribution
Report size
40-160
EV-concentration
No
EV220208 2/2 Homo sapiens HUVEC Filtration
dUC
Hosseinkhani B 2018 56%

Study summary

Full title
All authors
Hosseinkhani B, Kuypers S, van den Akker NMS, Molin DGM, Michiels L
Journal
Front Immunol
Abstract
Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial c (show more...)Extracellular vesicles (EV) mediated intercellular communication between monocytes and endothelial cells (EC) might play a major role in vascular inflammation and atherosclerotic plaque formation during cardiovascular diseases (CVD). While critical involvement of small (exosomes) and large EV (microvesicles) in CVD has recently been appreciated, the pro- and/or anti-inflammatory impact of a bulk EV (exosomes + microvesicles) on vascular cell function as well as their inflammatory capacity are poorly defined. This study aims to unravel the immunomodulatory content of EV bulk derived from control (uEV) and TNF-α induced inflamed endothelial cells (tEV) and to define their capacity to affect the inflammatory status of recipients monocytes (THP-1) and endothelial cells (HUVEC) . Here, we show that EV derived from inflamed vascular EC were readily taken up by THP-1 and HUVEC. Human inflammation antibody array together with ELISA revealed that tEV contain a pro-inflammatory profile with chemotactic mediators, including intercellular adhesion molecule (ICAM)-1, CCL-2, IL-6, IL-8, CXCL-10, CCL-5, and TNF-α as compared to uEV. In addition, EV may mediate a selective transfer of functional inflammatory mediators to their target cells and modulate them toward either pro-inflammatory (HUVEC) or anti/pro-inflammatory (THP-1) mode. Accordingly, the expression of pro-inflammatory markers (IL-6, IL-8, and ICAM-1) in tEV-treated HUVEC was increased. In the case of THP-1, EC-EV do induce a mixed of pro- and anti-inflammatory response as indicated by the elevated expression of ICAM-1, CCL-4, CCL-5, and CXCL-10 proteins. At the functional level, EC-EV mediated inflammation and promoted the adhesion and migration of THP-1. Taken together, our findings proved that the EV released from inflamed EC were enriched with a cocktail of inflammatory markers, chemokines, and cytokines which are able to establish a targeted cross-talk between EC and monocytes and reprogramming them toward a pro- or anti-inflammatory phenotypes. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
TNF-alpha
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
dUC
Protein markers
EV: CD63/ ICAM-1/ CD9/ IL-1B, IL-6, IL-8, ICAM-1, CCL-2, CCL-4, CCL-5, CCL-10, TNF-alpha/ GM-CSF/ IL-6/ IL-8/ TIMP2/ ICAM-1/ IL6-R/ CXCL10/ CCL-2/ CCL-5/ TNF-alpha/ TNF R/ PDGF-BB
non-EV: GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Other
Name other separation method
dUC
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ ICAM-1/ ?-Actin
Not detected contaminants
GM130
ELISA
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
IL-1B, IL-6, IL-8, ICAM-1, CCL-2, CCL-4, CCL-5, CCL-10, TNF-alpha
Other 1
ELISA
Detected EV-associated proteins
GM-CSF/ IL-6/ IL-8/ TIMP2/ ICAM-1/ IL6-R/ CXCL10/ CCL-2/ CCL-5/ TNF-alpha/ TNF R/ PDGF-BB
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV210501 1/2 Homo sapiens MCF-7 (d)(U)C Zhai LY 2018 56%

Study summary

Full title
All authors
Zhai LY, Li MX, Pan WL, Chen Y, Li MM, Pang JX, Zheng L, Chen JX, Duan WJ
Journal
ACS Appl Mater Interfaces
Abstract
Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, (show more...)Breast cancer is the second cause of cancer mortality in women globally. Early detection, treatment, and metastasis monitoring are of great importance to favorable prognosis. Although conventional diagnostic methods, such as breast X-ray mammography and image positioning biopsy, are accurate, they could cause radioactive or invasive damage to patients. Liquid biopsy as a noninvasive method is convenient for repeated sampling in clinical cancer prognostic, metastatic evaluation, and relapse monitoring. MicroRNAs encased in exosomes circulating in biofluids are promising candidate cancer biomarkers because of their cancer-specific expression profiles. Here, we report an in situ detection of microRNA-1246 (miR-1246) in human plasma exosomes as breast cancer biomarker by a nucleic acid functionalized Au nanoflare probe. Needing neither time-consuming and costly isolation of exosomes from the plasma sample nor transfection means, the Au nanoflare probe can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting miR-1246. Only 40 μL of plasma is needed to incubate 4 h with the probe, giving signal sensitive enough to distinguish samples of breast cancer to normal control. Using plasma miR-1246 level detected by our assay as a marker, we differentiated 46 breast cancer patients from 28 healthy controls with 100% sensitivity and 92.9% specificity at the best cutoff. This simple, accurate, sensitive, and cost-effective liquid biopsy by the Au nanoflare probe is potent to be developed as a noninvasive breast cancer diagnostic assay for clinical adaption. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ CD63
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development/Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MCF-7
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: time (min)
80
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ TSG101
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
Yes
Moment of Proteinase treatment
After
Proteinase type
Proteinase K
Proteinase concentration
64
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
4 U / mL
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
97.8
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
67.7
EV210389 1/1 Homo sapiens Urine dUC Liu Z 2018 56%

Study summary

Full title
All authors
Liu Z, Cauvi DM, Bernardino EMA, Lara B, Lizardo RE, Hawisher D, Bickler S, De Maio A
Journal
Cell Stress Chaperones
Abstract
Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential (show more...)Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential biomarkers for disease. They can be obtained from a variety of body fluids, particularly urine that is an ideal source because it can be obtained in great quantities, recurrently and with minimal intervention. However, the characterization of urine ECV is challenging because the preparation is usually contaminated with soluble proteins, such as uromodulin (UMOD) or Tamm-Horsfall glycoprotein that forms large extracellular filaments co-sedimenting with ECV. We developed a method to obtain human urine ECV free of UMOD by the addition of ZnSO prior to vesicle isolation by differential centrifugation. Treatment with ZnSO did not affect the size and concentration of the vesicle preparation and preserved the storage of the samples at low temperatures. We did not observe a variation in the number of vesicles isolated during different times of the day or different days between different donors. The glycoprotein pattern of urine ECV was characterized by binding to concanavalin A (Con A) and mass spectroscopy. Several markers were found, including dipeptidyl peptidase IV (CD26), vacuolar protein sorting factor 4A (VPS4A) and dipeptidase 1 (DPEP1), and galectin 3 binding protein (G3-BP). The levels of VPS4A and DPEP1 were similar in ECV preparations obtained from several donors of both sexes. Con A binding pattern and monosaccharide composition were also comparable between subjects. In summary, our method for the isolation of highly pure ECV derived from human urine is likely to help in the use of these vesicles as potential biomarkers. (hide)
EV-METRIC
56% (89th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
dUC
Protein markers
EV: DPEP1/ CD81/ G3BP/ CD9/ VPS4A
non-EV: Tamm-Horsfall protein
Proteomics
yes
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
1
Wash: time (min)
60
Wash: speed (g)
100000
Other
Name other separation method
dUC
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ VPS4A/ DPEP1/ G3BP/ CD81
Not detected contaminants
Tamm-Horsfall protein
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
136.9
EV concentration
Yes
Particle yield
as number of particles per milliliter of starting sample: 1.00e+10
EV200136 3/7 Homo sapiens Blood plasma DG
(d)(U)C
Filtration
Miranda, Jezid 2018 56%

Study summary

Full title
All authors
Jezid Miranda, Cristina Paules, Soumyalekshmi Nair, Andrew Lai, Carlos Palma, Katherin Scholz-Romero, Gregory E. Rice, Eduard Gratacos, Fatima Crispi, Carlos Salomon
Journal
Placenta
Abstract
Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta co (show more...)Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA). Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63þve and placental-type alkaline phosphatase (PLAP)þve antibodies, respectively. Results: Maternal concentrations of CD63þve and PLAPþve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAPþve ratio) and BW percentile, [rho ¼ 0.77 (95% CI: 0.57 to 0.89); p ¼ 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAPþve ratios in FGR compared to controls and SGA cases. Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function. (hide)
EV-METRIC
56% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Maternal; Fetus small for gestational age
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Filtration
Protein markers
EV: TSG101/ Flotillin1/ PLAP/ CD63
non-EV: Grp94
Proteomics
no
EV density (g/ml)
1.12-1.188g/ml
Show all info
Study aim
Function/Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
T-8100
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
10
Wash: time (min)
120
Wash: Rotor Type
T-8100
Wash: speed (g)
100000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Sample volume (mL)
0.5mL
Orientation
Bottom-up
Rotor type
T-8100
Speed (g)
100000
Duration (min)
1200
Fraction processing
Centrifugation
Pelleting: volume per fraction
0.05
Pelleting: duration (min)
120
Pelleting: rotor type
T-8100
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Detected EV-associated proteins
Flotillin1/ CD63/ TSG101
Not detected contaminants
Grp94
ELISA
Antibody details provided?
No
Detected EV-associated proteins
PLAP
Fluorescent NTA
Relevant measurements variables specified?
NA
Antibody details provided?
No
Detected EV-associated proteins
PLAP/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
82+/-18nm
EV concentration
Yes
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