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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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  • Experiments differ in Vesicle type/Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV140001 2/4 Homo sapiens Cell culture supernatant 0.2 µm filter
0.45 µm filter
Iodixanol-DG
UF
Van Deun J 2014 88%

Study summary

Full title
All authors
Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer an (show more...)Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers. (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + 0.45 µm filter + Iodixanol-DG + UF
Protein markers
EV: Alix/ CD63/ HSP90/ HSP70/ TSG101
non-EV: Cell organelle protein/ Ago2
Proteomics
no
EV density (g/ml)
1.094
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Density gradient
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Rotor type
SW32.1
Speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ HSP90/ HSP70/ TSG101
Detected contaminants
Cell organelle protein/ Ago2
Characterization: Particle analysis
NTA
EM
EM-type
immune EM
Image type
Wide-field
EV140104 3/3 Homo sapiens Milk Sucrose-DG Zonneveld MI 2014 75%

Study summary

Full title
All authors
Zonneveld MI, Brisson AR, van Herwijnen MJ, Tan S, van de Lest CH, Redegeld FA, Garssen J, Wauben MH, Nolte-'t Hoen EN
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an importan (show more...)Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at -80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system. (hide)
EV-METRIC
75% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Milk
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Sucrose-DG
Protein markers
EV: CD63/ CD9/ Flotilin1/ MHC2
non-EV: beta-casein/ lactoferrin
Proteomics
no
EV density (g/ml)
1.13-1.18
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Milk
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Lowest density fraction
0.4
Highest density fraction
2.5
Orientation
Top-down
Rotor type
SW60
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9/ Flotilin1/ MHC2
Not detected EV-associated proteins
lactoferrin
Detected contaminants
beta-casein
ELISA
Detected EV-associated proteins
MHC2
Characterization: Particle analysis
EM
EM-type
cryo EM
Image type
Close-up, Wide-field
EV140101 1/1 Gallus gallus Bile 0.2 µm filter
dUC
Sucrose-DG
Wang Y 2014 71%

Study summary

Full title
All authors
Wang Y, Wang G, Wang Z, Zhang H, Zhang L, Cheng Z
Journal
Biochem Cell Biol
Abstract
Exosomes, which are small membrane vesicles of endocytic origin, carry lipids, RNA/miRNAs, and prote (show more...)Exosomes, which are small membrane vesicles of endocytic origin, carry lipids, RNA/miRNAs, and proteins and have immune modulatory functions. In this study, we isolated exosomes from the bile of specific pathogen-free chickens, 42-43 days of age, by using an ultracentrifugation and filtration method. The density of the exosomes, isolated by sucrose gradient fractionation, was between 1.13 and 1.19 g/mL. Electron microscopic observation of the liver showed that exosomes were present in the space of Disse and bile canaliculus. Chicken biliary exosomes displayed typical saucer-shaped, rounded morphology. Using liquid chromatography mass spectrum methodology, 196 proteins, including exosomal markers and several unique proteins, were identified and compared with mouse biliary exosomes. Noteworthy, CCCH type zinc finger antiviral protein was found on chicken biliary exosomes never described before. Furthermore, our data show that chicken biliary exosomes promote the proliferation of CD4(+) and CD8(+) T cells and monocytes from liver. In addition, chicken biliary exosomes significantly inhibit avian leukosis virus subgroup J, which is an oncogenic retrovirus, from replicating in the DF-1 cell line. These data indicate that chicken biliary exosomes possess the capacity to influence the immune responses of lymphocytes and inhibit avian leukosis virus subgroup J (ALV-J). (hide)
EV-METRIC
71% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Bile
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG
Adj. k-factor
235.7 (pelleting) / 235.7 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.13-1.19
Show all info
Study aim
Function
Sample
Species
Gallus gallus
Sample Type
Bile
Isolation Method
Differential ultracentrifugation
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: time(min)
120
Pelleting: rotor type
P28S
Pelleting: adjusted k-factor
235.7
Wash: Rotor Type
P28S
Wash: adjusted k-factor
235.7
Density gradient
Lowest density fraction
8
Highest density fraction
45
Orientation
Top-down
Rotor type
P28S
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140063 1/1 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Burke M 2014 71%

Study summary

Full title
All authors
Burke M, Choksawangkarn W, Edwards N, Ostrand-Rosenberg S, Fenselau C
Journal
J Proteome Res
Abstract
Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit natur (show more...)Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit natural anti-tumor immunity and are an obstacle to anti-cancer immunotherapies. They mediate immune suppression through their production of proteins and soluble mediators that prevent the activation of tumor-reactive T lymphyocytes, polarize macrophages toward a tumor-promoting phenotype, and facilitate angiogenesis. The accumulation and suppressive potency of MDSC is regulated by inflammation within the tumor microenvironment. Recently exosomes have been proposed to act as intercellular communicators, carrying active proteins and other molecules between sender cells and receiver cells. In this report we describe the proteome of exosomes shed by MDSC induced in BALB/c mice by the 4T1 mammary carcinoma. Using bottom-up proteomics, we have identified 412 proteins. Spectral counting identified 63 proteins whose abundance was altered >2-fold in the inflammatory environment. The pro-inflammatory proteins S100A8 and S100A9, previously shown to be secreted by MDSC and to be chemotactic for MDSC, are abundant in MDSC-derived exosomes. Bioassays reveal that MDSC-derived exosomes polarize macrophages toward a tumor-promoting type 2 phenotype, in addition to possessing S100A8/A9 chemotactic activity. These results suggest that some of the tumor-promoting functions of MDSC are implemented by MDSC-shed exosomes. (hide)
EV-METRIC
71% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Adj. k-factor
276.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.2-1.3
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
1200
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
276.6
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140054 1/2 Homo sapiens Cell culture supernatant dUC
Sucrose-DG
Kranendonk ME 2014 67%

Study summary

Full title
All authors
Kranendonk ME, Visseren FL, van Balkom BW, Nolte-'t Hoen EN, van Herwaarden JA, de Jager W, Schipper HS, Brenkman AB, Verhaar MC, Wauben MH, Kalkhoven E
Journal
Obesity
Abstract
OBJECTIVE: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants (show more...)OBJECTIVE: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined. METHODS: EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes. RESULTS: Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR. CONCLUSIONS: A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Adj. k-factor
256 (pelleting)
Protein markers
EV: CD63/ CD9/ Flotilin1
non-EV: None
Proteomics
no
EV density (g/ml)
1.11-1.14
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
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: time(min)
120
Pelleting: rotor type
SW32;SW40;SW60
Pelleting: adjusted k-factor
256.0
Density gradient
Lowest density fraction
0.4
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9/ Flotilin1
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140001 1/4 Homo sapiens Cell culture supernatant 0.2 µm filter
0.45 µm filter
dUC
UF
Van Deun J 2014 67%

Study summary

Full title
All authors
Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer an (show more...)Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + 0.45 µm filter + dUC + UF
Adj. k-factor
138.6 (pelleting)
Protein markers
EV: Alix/ CD63/ HSP90/ HSP70/ TSG101
non-EV: Cell organelle protein/ Ago2
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
180
Pelleting: rotor type
SW55
Pelleting: adjusted k-factor
138.6
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ HSP90/ HSP70/ TSG101
Detected contaminants
Cell organelle protein/ Ago2
Characterization: Particle analysis
NTA
EM
EM-type
immune EM
Image type
Wide-field
EV140012 1/1 Homo sapiens Blood plasma 0.2 µm filter
dUC
Sucrose-DG
Salomon C 2014 67%

Study summary

Full title
All authors
Salomon C, Torres MJ, Kobayashi M, Scholz-Romero K, Sobrevia L, Dobierzewska A, Illanes SE, Mitchell MD, Rice GE
Journal
PLoS One
Abstract
Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a si (show more...)Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group) were obtained from non-pregnant and pregnant women in the first (FT, 6-12 weeks), second (ST, 22-24 weeks) and third (TT, 32-38 weeks) trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP), respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte). Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001). During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001). Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG
Adj. k-factor
53.28 (pelleting)
Protein markers
EV: CD63/ CD81/ CD9/ PLAP
non-EV: None
Proteomics
no
EV density (g/ml)
1.13-1.19
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultracentrifugation
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: time(min)
75
Pelleting: rotor type
T8100
Pelleting: adjusted k-factor
53.28
Wash: volume per pellet (ml)
30
Density gradient
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ PLAP
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9/ PLAP
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV140009 1/1 Homo sapiens Blood plasma 0.2 µm filter
dUC
SEC
Sucrose-DG (valid.)
Muller L 2014 67%

Study summary

Full title
All authors
Muller L, Hong CS, Stolz DB, Watkins SC, Whiteside TL
Journal
J Immunol Methods
Abstract
Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immuno (show more...)Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in ?g protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + SEC + Sucrose-DG (valid.)
Protein markers
EV: CD81/ CD9/ TSG101
non-EV: "GAPDH/ GPIIb/IIIa/ CD4/ CD69/ CD154"
Proteomics
no
EV density (g/ml)
1.150
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultracentrifugation
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: time(min)
120
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Top-down
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ CD9/ TSG101
Detected contaminants
"GAPDH/ GPIIb/IIIa/ CD4/ CD69/ CD154"
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140032 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Lee HD 2014 67%

Study summary

Full title
All authors
Lee HD, Kim YH, Kim DS
Journal
Eur J Immunol
Abstract
Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial fo (show more...)Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin ?1 in primary HUVECs. The internalized integrin ?1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin ?1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin ?1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin ?1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG (valid.)
Adj. k-factor
138.6 (pelleting)
Protein markers
EV: CD9/ TSG101/ ADAM10/ ADAM15/ GAPDH
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.13
TEM measurements
70-80
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
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: time(min)
60
Pelleting: rotor type
SW55
Pelleting: adjusted k-factor
138.6
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ TSG101/ ADAM10/ ADAM15/ GAPDH
ELISA
Detected EV-associated proteins
ADAM10/ ADAM15/ GAPDH
Characterization: Particle analysis
EM
EM-type
scanning EM
Image type
Close-up, Wide-field
EV140166 2/3 Mus musculus Pre-purified exosomes loaded with SPION5 Sucrose-DG (valid.) Hood JL 2014 67%

Study summary

Full title
All authors
Hood JL, Scott MJ, Wickline SA
Journal
Anal Biochem
Abstract
Development of exosome-based semisynthetic nanovesicles for diagnostic and therapeutic purposes requ (show more...)Development of exosome-based semisynthetic nanovesicles for diagnostic and therapeutic purposes requires novel approaches to load exosomes with cargo. Electroporation has previously been used to load exosomes with RNA. However, investigations into exosome colloidal stability following electroporation have not been considered. Herein, we report the development of a unique trehalose pulse media (TPM) that minimizes exosome aggregation following electroporation. Dynamic light scattering (DLS) and RNA absorbance were employed to determine the extent of exosome aggregation and electroextraction post electroporation in TPM compared to common PBS pulse media or sucrose pulse media (SPM). Use of TPM to disaggregate melanoma exosomes post electroporation was dependent on both exosome concentration and electric field strength. TPM maximized exosome dispersal post electroporation for both homogenous B16 melanoma and heterogeneous human serum-derived populations of exosomes. Moreover, TPM enabled heavy cargo loading of melanoma exosomes with 5nm superparamagnetic iron oxide nanoparticles (SPION5) while maintaining original exosome size and minimizing exosome aggregation as evidenced by transmission electron microscopy. Loading exosomes with SPION5 increased exosome density on sucrose gradients. This provides a simple, label-free means of enriching exogenously modified exosomes and introduces the potential for MRI-driven theranostic exosome investigations in vivo. (hide)
EV-METRIC
67% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Pre-purified exosomes loaded with SPION5
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Sucrose-DG (valid.)
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.16-1.19
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Pre-purified exosomes loaded with SPION5
Isolation Method
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140003 1/1 Homo sapiens Urine dUC
Sucrose-DG
Hogan MC 2014 67%

Study summary

Full title
All authors
Hogan MC, Johnson KL, Zenka RM, Charlesworth MC, Madden BJ, Mahoney DW, Oberg AL, Huang BQ, Leontovich AA, Nesbitt LL, Bakeberg JL, McCormick DJ, Bergen HR, Ward CJ
Journal
Kidney Int
Abstract
Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesi (show more...)Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40-200 nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm-Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV)-enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin-positive irregularly shaped membranous vesicles and podocin/podocalyxin-negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton, and Rho GDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases, and complement proteins involved in glomerular disease are in GMVs and some were only shed in the disease state (nephrin, TRPC6, INF2 and phospholipase A2 receptor). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome, and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease. (hide)
EV-METRIC
67% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
Membrane(-derived) vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Protein markers
EV: Podocin/ Polycystin1
non-EV: Tamm-Horsfall glycoprotein
Proteomics
yes
EV density (g/ml)
1.055
TEM measurements
91.4(median)
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Density gradient
Lowest density fraction
5
Highest density fraction
30
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Podocin/ Polycystin1
Detected contaminants
Tamm-Horsfall glycoprotein
ELISA
Detected EV-associated proteins
Podocin/ Polycystin1
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV140158 2/2 Homo sapiens Cell culture supernatant 0.1 µm filter
Sucrose-DG (valid.)
Fuhrmann G 2014 67%

Study summary

Full title
All authors
Fuhrmann G, Serio A, Mazo M, Nair R, Stevens MM
Journal
J Control Release
Abstract
Extracellular vesicles (EVs) are phospholipid-based particles endogenously produced by cells. Their (show more...)Extracellular vesicles (EVs) are phospholipid-based particles endogenously produced by cells. Their natural composition and selective cell interactions make them promising drug carriers. However, in order to harness their properties, efficient exogenous drug encapsulation methods need to be investigated. Here, EVs from various cellular origins (endothelial, cancer and stem cells) were produced and characterised for size and composition. Porphyrins of different hydrophobicities were employed as model drugs and encapsulated into EVs using various passive and active methods (electroporation, saponin, extrusion and dialysis). Hydrophobic compounds loaded very efficiently into EVs and at significantly higher amounts than into standard liposomes composed of phosphocholine and cholesterol using passive incubation. Moreover, loading into EVs significantly increased the cellular uptake by >60% and the photodynamic effect of hydrophobic porphyrins in vitro compared to free or liposome encapsulated drug. The active encapsulation techniques, with the saponin-assisted method in particular, allowed an up to 11 fold higher drug loading of hydrophilic porphyrins compared to passive methods. EVs loaded with hydrophilic porphyrins induced a stronger phototoxic effect than free drug in a cancer cell model. Our findings create a firm basis for the development of EVs as smart drug carriers based on straightforward and transferable methods. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.1 µm filter + Sucrose-DG (valid.)
Protein markers
EV: CD63/ CD40/ Phosphatidylserine
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.11
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Density gradient
Only used for validation of main results
1
Lowest density fraction
8
Highest density fraction
60
Orientation
Bottom-up
Filtration steps
0.2µm > x > 0.1µm
Western Blot
Detected EV-associated proteins
CD40/ Phosphatidylserine
ELISA
Detected EV-associated proteins
CD40/ Phosphatidylserine
Characterization: Particle analysis
DLS
NTA
Particle analysis: flow cytometry
EM
EM-type
transmission EM
Image type
Close-up
EV140002 1/1 Mus musculus Cell culture supernatant 0.2 µm filter
dUC
Forterre A 2014 67%

Study summary

Full title
All authors
Forterre A, Jalabert A, Berger E, Baudet M, Chikh K, Errazuriz E, De Larichaudy J, Chanon S, Weiss-Gayet M, Hesse AM, Record M, Geloen A, Lefai E, Vidal H, Couté Y, Rome S
Journal
PLoS One
Abstract
Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome ma (show more...)Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
Exosome-like vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
157.1 (pelleting)
Protein markers
EV: Alix/ CD81/ TSG101/ Beta-actin
non-EV: Cell organelle protein
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
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: time(min)
70
Pelleting: rotor type
50.2Ti
Pelleting: adjusted k-factor
157.1
Wash: volume per pellet (ml)
25
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD81/ TSG101/ Beta-actin
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Beta-actin
Characterization: Particle analysis
DLS
EM
EM-type
immune EM
Image type
Wide-field
EV140001 3/4 Homo sapiens Cell culture supernatant 0.2 µm filter
0.45 µm filter
Commercial
UF
Van Deun J 2014 63%

Study summary

Full title
All authors
Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer an (show more...)Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers. (hide)
EV-METRIC
63% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + 0.45 µm filter + Commercial + UF
Adj. k-factor
0 (pelleting)
Protein markers
EV: Alix/ CD63/ HSP90/ HSP70/ TSG101
non-EV: Cell organelle protein/ Ago2
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting: adjusted k-factor
NA
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ HSP90/ HSP70/ TSG101
Detected contaminants
Cell organelle protein/ Ago2
Characterization: Particle analysis
NTA
EM
EM-type
immune EM
Image type
Close-up
EV140001 4/4 Homo sapiens Cell culture supernatant 0.2 µm filter
0.45 µm filter
Commercial
UF
Van Deun J 2014 63%

Study summary

Full title
All authors
Van Deun J, Mestdagh P, Sormunen R, Cocquyt V, Vermaelen K, Vandesompele J, Bracke M, De Wever O, Hendrix A
Journal
J Extracell Vesicles
Abstract
Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer an (show more...)Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers. (hide)
EV-METRIC
63% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + 0.45 µm filter + Commercial + UF
Protein markers
EV: Alix/ CD63/ HSP90/ HSP70/ TSG101
non-EV: Cell organelle protein/ Ago2
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ HSP90/ HSP70/ TSG101
Detected contaminants
Cell organelle protein/ Ago2
Characterization: Particle analysis
NTA
EM
EM-type
immune EM
Image type
Close-up
EV140004 1/1 Ovis aries Uterine luminal fluid 0.2 µm filter
Commercial
Burns G 2014 63%

Study summary

Full title
All authors
Burns G, Brooks K, Wildung M, Navakanitworakul R, Christenson LK, Spencer TE
Journal
PLoS One
Abstract
Microvesicles and exosomes are nanoparticles released from cells and can contain small RNAs, mRNA an (show more...)Microvesicles and exosomes are nanoparticles released from cells and can contain small RNAs, mRNA and proteins that affect cells at distant sites. In sheep, endogenous beta retroviruses (enJSRVs) are expressed in the endometrial epithelia of the uterus and can be transferred to the conceptus trophectoderm. One potential mechanism of enJSRVs transfer from the uterus to the conceptus is via exosomes/microvesicles. Therefore, studies were conducted to evaluate exosomes in the uterine luminal fluid (ULF) of sheep. Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Transmission electron microscopy and nanoparticle tracking analysis found the isolates contained vesicles that ranged from 50 to 200 nm in diameter. The isolated extracellular vesicles were positive for two common markers of exosomes (CD63 and HSP70) by Western blot analysis. Proteins in the extracellular vesicles were determined by mass spectrometry and Western blot analysis. Extracellular vesicle RNA was analyzed for small RNAs by sequencing and enJSRVs RNA by RT-PCR. The ULF extracellular vesicles contained a large number of small RNAs and miRNAs including 81 conserved mature miRNAs. Cyclic and pregnant ULF extracellular vesicles contained enJSRVs env and gag RNAs that could be delivered to heterologous cells in vitro. These studies support the hypothesis that ULF extracellular vesicles can deliver enJSRVs RNA to the conceptus, which is important as enJSRVs regulate conceptus trophectoderm development. Importantly, these studies support the idea that extracellular vesicles containing select miRNAs, RNAs and proteins are present in the ULF and likely have a biological role in conceptus-endometrial interactions important for the establishment and maintenance of pregnancy. (hide)
EV-METRIC
63% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Uterine luminal fluid
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Commercial
Protein markers
EV: CD63/ HSP70
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Ovis aries
Sample Type
Uterine luminal fluid
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140111 2/2 Homo sapiens Cell culture supernatant dUC Cypryk W 2014 57%

Study summary

Full title
All authors
Cypryk W, Ohman T, Eskelinen EL, Matikainen S, Nyman TA
Journal
J Proteome Res
Abstract
Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially (show more...)Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially to immunologically compromised people. Fungi express a variety of pathogen-associated molecular patterns on their surface including ?-glucans, which are important immunostimulatory components of fungal cell walls. During stimulatory conditions of infection and colonization, besides intensive intracellular response, human cells actively communicate on the intercellular level by secreting proteins and other biomolecules with several mechanisms. Vesicular secretion remains one of the most important paths for the proteins to exit the cell. Here, we have used high-throughput quantitative proteomics combined with bioinformatics to characterize and quantify vesicle-mediated protein release from ?-glucan-stimulated human macrophages differentiated in vitro from primary blood monocytes. We show that ?-glucan stimulation induces vesicle-mediated protein secretion. Proteomic study identified 540 distinct proteins from the vesicles, and the identified proteins show a proteomic signature characteristic for their cellular origin. Importantly, we identified several receptors, including cation-dependent mannose-6-phosphate receptor, macrophage scavenger receptor, and P2X7 receptor, that have not been identified from vesicles before. Proteomic data together with detailed pathway and network analysis showed that integrins and their cytoplasmic cargo proteins are highly abundant in extracellular vesicles released upon ?-glucan stimulation. In conclusion, the present data provides a solid basis for further studies on the functional role of vesicular protein secretion upon fungal infection. (hide)
EV-METRIC
57% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
138.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
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: time(min)
60
Pelleting: rotor type
SW55
Pelleting: adjusted k-factor
138.6
Wash: volume per pellet (ml)
5
Characterization: Protein analysis
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV140046 1/1 Bacillus subtilis Bacteria 0.2 µm filter
0.8 µm filter
dUC
Iodixanol-DG (valid.)
UF
Brown L 2014 57%

Study summary

Full title
All authors
Brown L, Kessler A, Cabezas-Sanchez P, Luque-Garcia JL, Casadevall A
Journal
Mol Microbiol
Abstract
Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the abse (show more...)Previously, extracellular vesicle production in Gram-positive bacteria was dismissed due to the absence of an outer membrane, where Gram-negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram-positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harbouring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram-positive bacteria. We also identify a new mechanism of action for surfactin. (hide)
EV-METRIC
57% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Bacteria
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + 0.8 µm filter + dUC + Iodixanol-DG (valid.) + UF
Protein markers
EV: None
non-EV: None
Proteomics
yes
TEM measurements
137.69999999999999
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Bacillus subtilis
Sample Type
Bacteria
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
10
Highest density fraction
30
Orientation
Bottom-up
Filtration steps
> 0.45 µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV140106 1/2 Prochlorococcus Bacteria 0.2 µm filter
dUC
Iodixanol-DG (valid.)
UF
Biller SJ 2014 57%

Study summary

Full title
All authors
Biller SJ, Schubotz F, Roggensack SE, Thompson AW, Summons RE, Chisholm SW
Journal
Science
Abstract
Many heterotrophic bacteria are known to release extracellular vesicles, facilitating interactions b (show more...)Many heterotrophic bacteria are known to release extracellular vesicles, facilitating interactions between cells and their environment from a distance. Vesicle production has not been described in photoautotrophs, however, and the prevalence and characteristics of vesicles in natural ecosystems is unknown. Here, we report that cultures of Prochlorococcus, a numerically dominant marine cyanobacterium, continuously release lipid vesicles containing proteins, DNA, and RNA. We also show that vesicles carrying DNA from diverse bacteria are abundant in coastal and open-ocean seawater samples. Prochlorococcus vesicles can support the growth of heterotrophic bacterial cultures, which implicates these structures in marine carbon flux. The ability of vesicles to deliver diverse compounds in discrete packages adds another layer of complexity to the flow of information, energy, and biomolecules in marine microbial communities. (hide)
EV-METRIC
57% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Bacteria
Focus vesicles
Bacterial vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Iodixanol-DG (valid.) + UF
Adj. k-factor
256 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Prochlorococcus
Sample Type
Bacteria
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW32
Pelleting: adjusted k-factor
256.0
Density gradient
Only used for validation of main results
1
Highest density fraction
40
Orientation
Bottom-up
Rotor type
SW60
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ cryo EM/ scanning EM
Image type
Wide-field
EV140106 2/2 Prochlorococcus Coastal water 0.2 µm filter
dUC
Iodixanol-DG (valid.)
UF
Biller SJ 2014 57%

Study summary

Full title
All authors
Biller SJ, Schubotz F, Roggensack SE, Thompson AW, Summons RE, Chisholm SW
Journal
Science
Abstract
Many heterotrophic bacteria are known to release extracellular vesicles, facilitating interactions b (show more...)Many heterotrophic bacteria are known to release extracellular vesicles, facilitating interactions between cells and their environment from a distance. Vesicle production has not been described in photoautotrophs, however, and the prevalence and characteristics of vesicles in natural ecosystems is unknown. Here, we report that cultures of Prochlorococcus, a numerically dominant marine cyanobacterium, continuously release lipid vesicles containing proteins, DNA, and RNA. We also show that vesicles carrying DNA from diverse bacteria are abundant in coastal and open-ocean seawater samples. Prochlorococcus vesicles can support the growth of heterotrophic bacterial cultures, which implicates these structures in marine carbon flux. The ability of vesicles to deliver diverse compounds in discrete packages adds another layer of complexity to the flow of information, energy, and biomolecules in marine microbial communities. (hide)
EV-METRIC
57% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Coastal water
Focus vesicles
Bacterial vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Iodixanol-DG (valid.) + UF
Adj. k-factor
256 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Prochlorococcus
Sample Type
Coastal water
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW32
Pelleting: adjusted k-factor
256.0
Density gradient
Only used for validation of main results
1
Highest density fraction
40
Orientation
Bottom-up
Rotor type
SW60
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ cryo EM/ scanning EM
Image type
Wide-field
EV140018 1/2 Homo sapiens
Gallus gallus
Cell culture supernatant dUC
Sucrose-DG (valid.)
Vyas N 2014 56%

Study summary

Full title
All authors
Vyas N, Walvekar A, Tate D, Lakshmanan V, Bansal D, Lo Cicero A, Raposo G, Palakodeti D, Dhawan J
Journal
Sci Rep
Abstract
Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tiss (show more...)Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3? and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: CD9/ TSG101
non-EV: Cell organelle protein/ "Flotillin1/ Flotillin2"
Proteomics
yes
EV density (g/ml)
1.120
Show all info
Study aim
Function
Sample
Species
Homo sapiens / Gallus gallus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
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
Equal to or above 150,000 g
Pelleting: time(min)
90
Density gradient
Only used for validation of main results
1
Lowest density fraction
10
Highest density fraction
85
Orientation
Top-down
Speed (g)
450000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Detected contaminants
Cell organelle protein/ "Flotillin1/ Flotillin2"
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV140018 2/2 Homo sapiens
Gallus gallus
Cell culture supernatant dUC
Sucrose-DG (valid.)
Vyas N 2014 56%

Study summary

Full title
All authors
Vyas N, Walvekar A, Tate D, Lakshmanan V, Bansal D, Lo Cicero A, Raposo G, Palakodeti D, Dhawan J
Journal
Sci Rep
Abstract
Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tiss (show more...)Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3? and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: CD9/ Flotilin1/ TSG101/ Flotillin2
non-EV: Cell organelle protein
Proteomics
yes
EV density (g/ml)
1.120
Show all info
Study aim
Function
Sample
Species
Homo sapiens / Gallus gallus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
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: time(min)
90
Density gradient
Only used for validation of main results
1
Lowest density fraction
10
Highest density fraction
85
Orientation
Top-down
Speed (g)
150000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ Flotilin1/ TSG101/ Flotillin2
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Flotillin2
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV140050 1/1 Streptococcus pneumoniae Bacteria 0.2 µm filter
dUC
Iodixanol-DG (valid.)
SEC
Olaya-Abril A 2014 56%

Study summary

Full title
All authors
Olaya-Abril A, Prados-Rosales R, McConnell MJ, Martín-Peña R, González-Reyes JA, Jiménez-Munguía I, Gómez-Gascón L, Fernández J, Luque-García JL, García-Lidón C, Estévez H, Pachón J, Obando I, Casadevall A, Pirofski LA, Rodríguez-Ortega MJ
Journal
J Proteomics
Abstract
Extracellular vesicles are produced by many pathogenic microorganisms and have varied functions that (show more...)Extracellular vesicles are produced by many pathogenic microorganisms and have varied functions that include secretion and release of microbial factors, which contribute to virulence. Very little is known about vesicle production by Gram-positive bacteria, as well as their biogenesis and release mechanisms. In this work, we demonstrate the active production of vesicles by Streptococcus pneumoniae from the plasma membrane, rather than being a product from cell lysis. We biochemically characterized them by proteomics and fatty acid analysis, showing that these vesicles and the plasma membrane resemble in essential aspects, but have some differences: vesicles are more enriched in lipoproteins and short-chain fatty acids. We also demonstrate that these vesicles act as carriers of surface proteins and virulence factors. They are also highly immunoreactive against human sera and induce immune responses that protect against infection. Overall, this work provides insights into the biology of this important Gram-positive human pathogen and the role of extracellular vesicles in clinical applications.BIOLOGICAL SIGNIFICANCE: Pneumococcus is one of the leading causes of bacterial pneumonia worldwide in children and the elderly, being responsible for high morbidity and mortality rates in developing countries. The augment of pneumococcal disease in developed countries has raised major public health concern, since the difficulties to treat these infections due to increasing antibiotic resistance. Vaccination is still the best way to combat pneumococcal infections. One of the mechanisms that bacterial pathogens use to combat the defense responses of invaded hosts is the production and release of extracellular vesicles derived from the outer surface. Little is known about this phenomenon in Gram-positives. We show that pneumococcus produces membrane-derived vesicles particularly enriched in lipoproteins. We also show the utility of pneumococcal vesicles as a new type of vaccine, as they induce protection in immunized mice against infection with a virulent strain. This work will contribute to understand the role of these structures in important biological processes such as host-pathogen interactions and prevention of human disease. (hide)
EV-METRIC
56% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Bacteria
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Iodixanol-DG (valid.) + SEC
Protein markers
EV: "MalX/ Pneumococcal surface protein A"
non-EV: Pneumolysin
Proteomics
yes
TEM measurements
20-75
Show all info
Study aim
Function
Sample
Species
Streptococcus pneumoniae
Sample Type
Bacteria
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Density gradient
Only used for validation of main results
1
Lowest density fraction
5
Highest density fraction
35
Orientation
Bottom-up
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
"MalX/ Pneumococcal surface protein A"
Detected contaminants
Pneumolysin
ELISA
Detected EV-associated proteins
"MalX/ Pneumococcal surface protein A"
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM/ scanning EM
EV140115 1/4 Homo sapiens Cell culture supernatant dUC
UF
Lozito TP 2014 56%

Study summary

Full title
All authors
Lozito TP, Tuan RS
Journal
J Cell Mol Med
Abstract
Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSC (show more...)Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell-derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non-MP secreted factors (Sup) were isolated from serum-free medium conditioned by human microvascular ECs (HMEC-1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP-containing MPs were isolated from cells transduced with CMV-GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP-MPs, but not free GFP. Thus, only MP-associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP-1, MMP-3, CCL-2/MCP-1 and IL-6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF-?B signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
microparticles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + UF
Adj. k-factor
276.6 (pelleting)
Protein markers
EV: Calnexin/ Caveolin/ GAPDH
non-EV: Cell organelle protein/ "LAMP2/ Rab5"
Proteomics
no
TEM measurements
50-115
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
276.6
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Calnexin/ Caveolin/ GAPDH
Detected contaminants
Cell organelle protein/ "LAMP2/ Rab5"
ELISA
Detected EV-associated proteins
Calnexin/ Caveolin/ GAPDH
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV140017 1/1 Homo sapiens Serum dUC
Sucrose-DG
Di Noto G 2014 56%

Study summary

Full title
All authors
Di Noto G, Chiarini M, Paolini L, Mazzoldi EL, Giustini V, Radeghieri A, Caimi L, Ricotta D
Journal
Front Immunol
Abstract
Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistenc (show more...)Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. Monoclonal plasma cells often secrete high amounts of immunoglobulin free light chains (FLCs) that could induce tissue damage. Recently, we showed that FLCs are internalized in endothelial and myocardial cell lines and secreted in extracellular vesicles (EVs). MM serum derived EVs presented phenotypic differences if compared with monoclonal gammopathy of undetermined significance (MGUS) serum derived EVs suggesting their involvement in MM pathogenesis or progression. To investigate the effect of circulating EVs on endothelial and myocardial cells, we purified MM and MGUS serum derived EVs with differential ultracentrifugation protocols and tested their biological activity. We found that MM and MGUS EVs induced different proliferation and internalization rates in endothelial and myocardial cells, thus we tried to find specific targets in MM EVs docking and processing. Pre-treatment of EVs with anti-FLCs antibodies or heparin blocked the MM EVs uptake, highlighting that FLCs and glycosaminoglycans are involved. Indeed, only MM EVs exposure induced a strong nuclear factor kappa B nuclear translocation that was completely abolished after anti-FLCs antibodies and heparin pre-treatment. The protein tyrosine kinase c-src is present on MM circulating EVs and redistributes to the cell plasma membrane after MM EVs exposure. The anti-FLCs antibodies and heparin pre-treatments were able to block the intracellular re-distribution of the c-src kinase and the subsequent c-src kinase containing EVs production. Our results open new insights in EVs cellular biology and in MM therapeutic and diagnostic approaches. (hide)
EV-METRIC
56% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Protein markers
EV: CD63/ HSP70/ TSG101/ Annexin5
non-EV: None
Proteomics
no
EV density (g/ml)
1.11-1.22
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Serum
Isolation Method
Differential ultracentrifugation
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: time(min)
120
Density gradient
Lowest density fraction
15
Highest density fraction
60
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ HSP70/ TSG101/ Annexin5
ELISA
Detected EV-associated proteins
Annexin5
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
scanning EM/ atomic force EM
Image type
Close-up, Wide-field
EV140104 1/3 Mus musculus Cell culture supernatant dUC
Sucrose-DG
Zonneveld MI 2014 56%

Study summary

Full title
All authors
Zonneveld MI, Brisson AR, van Herwijnen MJ, Tan S, van de Lest CH, Redegeld FA, Garssen J, Wauben MH, Nolte-'t Hoen EN
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an importan (show more...)Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at -80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: CD63/ CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.13-1.18
Show all info
Study aim
Technical
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
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: time(min)
65
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
253.9
Density gradient
Lowest density fraction
0.4
Highest density fraction
2.5
Orientation
Top-down
Rotor type
SW60
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9
Characterization: Particle analysis
EV140016 1/2 Homo sapiens Saliva dUC Zlotogorski-Hurvitz A 2014 56%

Study summary

Full title
All authors
Zlotogorski-Hurvitz A, Dayan D, Chaushu G, Korvala J, Salo T, Sormunen R, Vered M
Journal
J Histochem Cytochem
Abstract
ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for us (show more...)ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC. (hide)
EV-METRIC
56% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Saliva
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
34.54 (pelleting)
Protein markers
EV: CD63/ CD81/ CD9/ Beta-actin
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Saliva
Isolation Method
Differential ultracentrifugation
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: time(min)
180
Pelleting: rotor type
TLA120.2
Pelleting: adjusted k-factor
34.54
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ Beta-actin
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9/ Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM/ atomic force EM
Image type
Close-up, Wide-field
EV140013 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG
Smyth TJ 2014 56%

Study summary

Full title
All authors
Smyth TJ, Redzic JS, Graner MW, Anchordoquy TJ
Journal
Biochim Biophys Acta Biomembranes
Abstract
Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. Th (show more...)Small endogenous vesicles called exosomes are beginning to be explored as drug delivery vehicles. The in vivo targets of exosomes are poorly understood; however, they are believed to be important in cell-to-cell communication and may play a prominent role in cancer metastasis. We aimed to elucidate whether cancer derived exosomes can be used as drug delivery vehicles that innately target tumors over normal tissue. Our in vitro results suggest that while there is some specificity towards cancer cells over immortalized cells, it is unclear if the difference is sufficient to achieve precise in vivo targeting. Additionally, we found that exosomes associate with their cellular targets to a significantly greater extent (>10-fold) than liposomes of a similar size. Studies on the association of liposomes mimicking the unique lipid content of exosomes revealed that the lipid composition contributes significantly to cellular adherence/internalization. Cleavage of exosome surface proteins yielded exosomes exhibiting reduced association with their cellular targets, demonstrating the importance of proteins in binding/internalization. Furthermore, although acidic conditions are known to augment the metastatic potential of tumors, we found that cells cultured at low pH released exosomes with significantly less potential for cellular association than cells cultured at physiological pH. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Protein markers
EV: CD63/ CD9/ HSP90/ HSP70
non-EV: Cell organelle protein
Proteomics
yes
EV density (g/ml)
1.18
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
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: time(min)
600
Density gradient
Lowest density fraction
12
Highest density fraction
50
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9/ HSP90/ HSP70
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV140039 1/1 Homo sapiens Blood plasma 0.2 µm filter
dUC
SEC
Sucrose-DG (valid.)
Schuler PJ 2014 56%

Study summary

Full title
All authors
Schuler PJ, Saze Z, Hong CS, Muller L, Gillespie DG, Cheng D, Harasymczuk M, Mandapathil M, Lang S, Jackson EK, Whiteside TL
Journal
Clin Exp Immunol
Abstract
While murine CD4(+) CD39(+) regulatory T cells (T(reg)) co-express CD73 and hydrolyze exogenous (e) (show more...)While murine CD4(+) CD39(+) regulatory T cells (T(reg)) co-express CD73 and hydrolyze exogenous (e) adenosine triphosphate (ATP) to immunosuppressive adenosine (ADO), surface co-expression of CD73 on human circulating CD4(+) CD39(+) T(reg) is rare. Therefore, the ability of human T(reg) to produce and utilize ADO for suppression remains unclear. Using mass spectrometry, we measured nucleoside production by subsets of human CD4(+) CD39(+) and CD4(+) CD39(-)CD73(+) T cells or CD19(+) B cells isolated from blood of 30 volunteers and 14 cancer patients. CD39 and CD73 expression was evaluated by flow cytometry, Western blots, confocal microscopy or reverse transcription-polymerase chain reaction (RT-PCR). Circulating CD4(+) CD39(+) T(reg) which hydrolyzed eATP to 5'-AMP contained few intracytoplasmic granules and had low CD73 mRNA levels. Only ?1% of these T(reg) were CD39(+) CD73(+) . In contrast, CD4(+) CD39(neg) CD73(+) T cells contained numerous CD73(+) granules in the cytoplasm and strongly expressed surface CD73. In vitro-generated T(reg) (Tr1) and most B cells were CD39(+) CD73(+) . All these CD73(+) T cell subsets and B cells hydrolyzed 5'-AMP to ADO. Exosomes isolated from plasma of normal control (NC) or cancer patients carried enzymatically active CD39 and CD73(+) and, when supplied with eATP, hydrolyzed it to ADO. Only CD4(+) CD39(+) T(reg) co-incubated with CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes produced ADO. Thus, contact with membrane-tethered CD73 was sufficient for ADO production by CD4(+) CD39(+) T(reg). In microenvironments containing CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes, CD73 is readily available to CD4(+) CD39(+) CD73(neg) T(reg) for the production of immunosuppressive ADO. (hide)
EV-METRIC
56% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + SEC + Sucrose-DG (valid.)
Protein markers
EV: CD81/ CD39/ CD73
non-EV: None
Proteomics
no
EV density (g/ml)
1.15-1.2
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultracentrifugation
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: time(min)
180
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.2
Highest density fraction
2.5
Orientation
Top-down
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ CD39/ CD73
ELISA
Detected EV-associated proteins
CD39/ CD73
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up
EV140094 1/1 Homo sapiens Blood plasma 0.2 µm filter
dUC
Sucrose-DG
Sarker S 2014 56%

Study summary

Full title
All authors
Sarker S, Scholz-Romero K, Perez A, Illanes SE, Mitchell MD, Rice GE, Salomon C
Journal
J Transl Med
Abstract
BACKGROUND: Human placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulat (show more...)BACKGROUND: Human placenta releases specific nanovesicles (i.e. exosomes) into the maternal circulation during pregnancy, however, the presence of placenta-derived exosomes in maternal blood during early pregnancy remains to be established. The aim of this study was to characterise gestational age related changes in the concentration of placenta-derived exosomes during the first trimester of pregnancy (i.e. from 6 to 12 weeks) in plasma from women with normal pregnancies. METHODS: A time-series experimental design was used to establish pregnancy-associated changes in maternal plasma exosome concentrations during the first trimester. A series of plasma were collected from normal healthy women (10 patients) at 6, 7, 8, 9, 10, 11 and 12 weeks of gestation (n = 70). We measured the stability of these vesicles by quantifying and observing their protein and miRNA contents after the freeze/thawing processes. Exosomes were isolated by differential and buoyant density centrifugation using a sucrose continuous gradient and characterised by their size distribution and morphology using the nanoparticles tracking analysis (NTA; Nanosight™) and electron microscopy (EM), respectively. The total number of exosomes and placenta-derived exosomes were determined by quantifying the immunoreactive exosomal marker, CD63 and a placenta-specific marker (Placental Alkaline Phosphatase PLAP). RESULTS: These nanoparticles are extraordinarily stable. There is no significant decline in their yield with the freeze/thawing processes or change in their EM morphology. NTA identified the presence of 50-150 nm spherical vesicles in maternal plasma as early as 6 weeks of pregnancy. The number of exosomes in maternal circulation increased significantly (ANOVA, p = 0.002) with the progression of pregnancy (from 6 to 12 weeks). The concentration of placenta-derived exosomes in maternal plasma (i.e. PLAP+) increased progressively with gestational age, from 6 weeks 70.6 ± 5.7 pg/ml to 12 weeks 117.5 ± 13.4 pg/ml. Regression analysis showed that weeks is a factor that explains for >70% of the observed variation in plasma exosomal PLAP concentration while the total exosome number only explains 20%. CONCLUSIONS: During normal healthy pregnancy, the number of exosomes present in the maternal plasma increased significantly with gestational age across the first trimester of pregnancy. This study is a baseline that provides an ideal starting point for developing early detection method for women who subsequently develop pregnancy complications, clinically detected during the second trimester. Early detection of women at risk of pregnancy complications would provide an opportunity to develop and evaluate appropriate intervention strategies to limit acute adverse sequel. (hide)
EV-METRIC
56% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG
Adj. k-factor
53.28 (pelleting) / 53.28 (washing)
Protein markers
EV: CD63
non-EV: None
Proteomics
yes
EV density (g/ml)
1.12-1.2
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultracentrifugation
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: time(min)
120
Pelleting: rotor type
T8100
Pelleting: adjusted k-factor
53.28
Wash: volume per pellet (ml)
5
Wash: Rotor Type
T8100
Wash: adjusted k-factor
53.28
Density gradient
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63
ELISA
Detected EV-associated proteins
CD63
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV140090 1/1 Mus musculus Serum 0.2 µm filter
dUC
Iodixanol-DG
Phoonsawat W 2014 56%

Study summary

Full title
All authors
Phoonsawat W, Aoki-Yoshida A, Tsuruta T, Sonoyama K
Journal
Biochem Biophys Res Commun
Abstract
Exosomes are membrane vesicles 30-120 nm in diameter that are released by many cell types and carry (show more...)Exosomes are membrane vesicles 30-120 nm in diameter that are released by many cell types and carry a cargo of proteins, lipids, mRNA, and microRNA. Cultured adipocytes reportedly release exosomes that may play a role in cell-to-cell communication during the development of metabolic diseases. However, the characteristics and function of exosomes released from adipocytes in vivo remain to be elucidated. Clearly, adipocyte-derived exosomes could exist in the circulation and may be associated with adipocyte-specific proteins such as adipocytokines. We isolated exosomes from serum of mice by differential centrifugation and analyzed adiponectin, leptin, and resistin in the exosome fraction. Western blotting detected adiponectin but no leptin and only trace amounts of resistin in the exosome fraction. The adiponectin signal in the exosome fraction was decreased by proteinase K treatment and completely quenched by a combination of proteinase K and Triton X-100. Quantitative ELISA showed that the exosome fraction contains considerable amounts of adiponectin, but not leptin or resistin. The concentration of adiponectin in the serum and the ratio of adiponectin to total protein in the exosome fraction were lower in obese mice than in lean mice. These results suggest that a portion of adiponectin exists as a transmembrane protein in the exosomes in mouse serum. We propose adiponectin as a marker of exosomes released from adipocytes in vivo. (hide)
EV-METRIC
56% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Iodixanol-DG
Protein markers
EV: CD63
non-EV: None
Proteomics
no
EV density (g/ml)
1.17
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Serum
Isolation Method
Differential ultracentrifugation
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: time(min)
120
Wash: volume per pellet (ml)
1
Density gradient
Lowest density fraction
6
Highest density fraction
50
Orientation
Top-down
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63
ELISA
Detected EV-associated proteins
CD63
Characterization: Particle analysis
EV140010 1/1 Homo sapiens Cell culture supernatant dUC Ostenfeld MS 2014 56%

Study summary

Full title
All authors
Ostenfeld MS, Jeppesen DK, Laurberg JR, Boysen AT, Bramsen JB, Primdal-Bengtson B, Hendrix A, Lamy P, Dagnaes-Hansen F, Rasmussen MH, Bui KH, Fristrup N, Christensen EI, Nordentoft I, Morth JP, Jensen JB, Pedersen JS, Beck M, Theodorescu D, Borre M, Howard KA, Dyrskjøt L, Ørntoft TF
Journal
Cancer Res
Abstract
Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cancer, (show more...)Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cancer, exosome secretion has been implicated in tumor growth and metastatic spread. In this study, we explored the possibility that exosomal pathways might discard tumor-suppressor miRNA that restricts metastatic progression. Secreted miRNA characterized from isogenic bladder carcinoma cell lines with differing metastatic potential were uncoupled from binding to target transcripts or the AGO2-miRISC complex. In metastatic cells, we observed a relative increase in secretion of miRNA with tumor-suppressor functions, including miR23b, miR224, and miR921. Ectopic expression of miR23b inhibited invasion, anoikis, angiogenesis, and pulmonary metastasis. Silencing of the exocytotic RAB family members RAB27A or RAB27B halted miR23b and miR921 secretion and reduced cellular invasion. Clinically, elevated levels of RAB27B expression were linked to poor prognosis in two independent cohorts of patients with bladder cancer. Moreover, highly exocytosed miRNA from metastatic cells, such as miR23b, were reduced in lymph node metastases compared with patient-matched primary tumors and were correlated with increments in miRNA-targeted RNA. Taken together, our results suggested that exosome-mediated secretion of tumor-suppressor miRNA is selected during tumor progression as a mechanism to coordinate activation of a metastatic cascade. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
156.9 (pelleting)
Protein markers
EV: CD63/ CD81/ HSP90/ TSG101/ Beta-actin
non-EV: Cell organelle protein/ Ago2
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
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: time(min)
120
Pelleting: rotor type
70Ti;Surespin630
Pelleting: adjusted k-factor
156.9
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ HSP90/ TSG101/ Beta-actin
Detected contaminants
Cell organelle protein/ Ago2
ELISA
Detected EV-associated proteins
Beta-actin
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV140035 1/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Melo SA 2014 56%

Study summary

Full title
All authors
Melo SA, Sugimoto H, O'Connell JT, Kato N, Villanueva A, Vidal A, Qiu L, Vitkin E, Perelman LT, Melo CA, Lucci A, Ivan C, Calin GA, Kalluri R
Journal
Cancer Cell
Abstract
Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that (show more...)Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
276.6 (pelleting) / 276.6 (washing)
Protein markers
EV: CD63/ CD9/ Flotilin1/ TSG101/ GAPDH/ Ago2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
276.6
Wash: Rotor Type
SW40
Wash: adjusted k-factor
276.6
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9/ Flotilin1/ TSG101/ GAPDH/ Ago2
ELISA
Detected EV-associated proteins
GAPDH/ Ago2
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
NTA
Particle analysis: flow cytometry
EV140081 2/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Other method
Im H 2014 56%

Study summary

Full title
All authors
Im H, Shao H, Park YI, Peterson VM, Castro CM, Weissleder R, Lee H
Journal
Nat Biotechnol
Abstract
Exosomes show potential for cancer diagnostics because they transport molecular contents of the cell (show more...)Exosomes show potential for cancer diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires extensive sample purification and labeling. Here we describe a label-free, high-throughput approach for quantitative analysis of exosomes. Our nano-plasmonic exosome (nPLEX) assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. We show that this approach offers improved sensitivity over previous methods, enables portable operation when integrated with miniaturized optics and allows retrieval of exosomes for further study. Using nPLEX to analyze ascites samples from ovarian cancer patients, we find that exosomes derived from ovarian cancer cells can be identified by their expression of CD24 and EpCAM, suggesting the potential of exosomes for diagnostics. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Other method
Protein markers
EV: CD63/ CD9/ Flotilin1/ HSP90/ HSP70/ Flotillin2
non-EV: Integrin-beta1/ Integrin-alpha5
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Filtration steps
0.22µm or 0.2µm
Other
Name other isolation method
Microfluidics
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9/ Flotilin1/ HSP90/ HSP70/ Flotillin2
Detected contaminants
Integrin-beta1/ Integrin-alpha5
ELISA
Detected EV-associated proteins
CD63/ CD9/ Flotilin1/ HSP90/ HSP70/ Flotillin2
Detected contaminants
Integrin-beta1/ Integrin-alpha5
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ scanning EM
Image type
Close-up, Wide-field
EV140080 2/2 Homo sapiens Urine dUC
Sucrose-DG (valid.)
Hogan MC 2014 56%

Study summary

Full title
All authors
Hogan MC, Bakeberg JL, Gainullin VG, Irazabal MV, Harmon AJ, Lieske JC, Charlesworth MC, Johnson KL, Madden BJ, Zenka RM, McCormick DJ, Sundsbak JL, Heyer CM, Torres VE, Harris PC, Ward CJ
Journal
J Am Soc Nephrol
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals (show more...)Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P<0.03). In samples from individuals with PKD1 mutations, levels of PC1 and PC2 were reduced to 54% (P<0.02) and 53% (P<0.001), respectively. Transmembrane protein 2 (TMEM2), a protein with homology to fibrocystin, was 2.1-fold higher in individuals with PKD1 mutations (P<0.03). The PC1/TMEM2 ratio correlated inversely with height-adjusted total kidney volume in the discovery cohort, and the ratio of PC1/TMEM2 or PC2/TMEM2 could be used to distinguish individuals with PKD1 mutations from controls in a confirmation cohort. In summary, results of this study suggest that a test measuring the urine exosomal PC1/TMEM2 or PC2/TMEM2 ratio may have utility in diagnosis and monitoring of polycystic kidney disease. Future studies will focus on increasing sample size and confirming these studies. The data were deposited in the ProteomeXchange (identifier PXD001075). (hide)
EV-METRIC
56% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Adj. k-factor
168.7 (pelleting)
Protein markers
EV: Alix/ Polycystin1/ Polycystin2/ Fibrocystin/ TMEM2
non-EV: None
Proteomics
yes
EV density (g/ml)
1.046-1.065
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
T647.5
Pelleting: adjusted k-factor
168.7
Density gradient
Only used for validation of main results
1
Lowest density fraction
5
Highest density fraction
30
Orientation
Bottom-up
Rotor type
Surespin
Speed (g)
200000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Polycystin1/ Polycystin2/ Fibrocystin/ TMEM2
ELISA
Detected EV-associated proteins
Polycystin1/ Polycystin2/ Fibrocystin/ TMEM2
Characterization: Particle analysis
EM
EV140005 1/1 Homo sapiens Urine 0.2 µm filter
dUC
Hiemstra TF 2014 56%

Study summary

Full title
All authors
Hiemstra TF, Charles PD, Gracia T, Hester SS, Gatto L, Al-Lamki R, Floto RA, Su Y, Skepper JN, Lilley KS, Karet Frankl FE
Journal
J Am Soc Nephrol
Abstract
Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocyt (show more...)Exosomes are small extracellular vesicles, approximately 50 nm in diameter, derived from the endocytic pathway and released by a variety of cell types. Recent data indicate a spectrum of exosomal functions, including RNA transfer, antigen presentation, modulation of apoptosis, and shedding of obsolete protein. Exosomes derived from all nephron segments are also present in human urine, where their function is unknown. Although one report suggested in vitro uptake of exosomes by renal cortical collecting duct cells, most studies of human urinary exosomes have focused on biomarker discovery rather than exosome function. Here, we report results from in-depth proteomic analyses and EM showing that normal human urinary exosomes are significantly enriched for innate immune proteins that include antimicrobial proteins and peptides and bacterial and viral receptors. Urinary exosomes, but not the prevalent soluble urinary protein uromodulin (Tamm-Horsfall protein), potently inhibited growth of pathogenic and commensal Escherichia coli and induced bacterial lysis. Bacterial killing depended on exosome structural integrity and occurred optimally at the acidic pH typical of urine from omnivorous humans. Thus, exosomes are innate immune effectors that contribute to host defense within the urinary tract. (hide)
EV-METRIC
56% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
89.26 (pelleting)
Protein markers
EV: CD63/ TSG101/ Alpha-enolase/ Podocin
non-EV: None
Proteomics
yes
TEM measurements
54.5+-14
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
135
Pelleting: rotor type
45Ti
Pelleting: adjusted k-factor
89.26
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ Alpha-enolase/ Podocin
ELISA
Detected EV-associated proteins
Alpha-enolase/ Podocin
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up
EV140025 1/1 Drosophila melanogaster Drosophila 0.2 µm filter
dUC
Sucrose-DG (valid.)
Gradilla AC 2014 56%

Study summary

Full title
All authors
Gradilla AC, González E, Seijo I, Andrés G, Bischoff M, González-Mendez L, Sánchez V, Callejo A, Ibáñez C, Guerra M, Ortigão-Farias JR, Sutherland JD, González M, Barrio R, Falcón-Pérez JM, Guerrero I
Journal
Nat Commun
Abstract
The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migrat (show more...)The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen. (hide)
EV-METRIC
56% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Drosophila
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG (valid.)
Protein markers
EV: HSP70/ TSG101/ Rab11/ Rab8/ Syntaxin
non-EV: ApoLII
Proteomics
no
EV density (g/ml)
1.12-1.19
Show all info
Study aim
Function
Sample
Species
Drosophila melanogaster
Sample Type
Drosophila
Isolation Method
Differential ultracentrifugation
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: time(min)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Rotor type
TLA110
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
HSP70/ TSG101/ Rab11/ Rab8/ Syntaxin
Detected contaminants
ApoLII
ELISA
Detected EV-associated proteins
Rab11/ Rab8/ Syntaxin
Characterization: Particle analysis
EV140056 1/1 Oryctolagus cuniculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Arellano-Anaya ZE 2014 56%

Study summary

Full title
All authors
Arellano-Anaya ZE, Huor A, Leblanc P, Lehmann S, Provansal M, Raposo G, Andréoletti O, Vilette D
Journal
Cell Mol Life Sci
Abstract
Cell-to-cell transfer of prions is a crucial step in the spreading of prion infection through infect (show more...)Cell-to-cell transfer of prions is a crucial step in the spreading of prion infection through infected tissue. At the cellular level, several distinct pathways including direct cell-cell contacts and release of various types of infectious extracellular vesicles have been described that may potentially lead to infection of naïve cells. The relative contribution of these pathways and whether they may vary depending on the prion strain and/or on the infected cell type are not yet known. In this study we used a single cell type (RK13) infected with three different prion strains. We showed that in each case, most of the extracellular prions resulted from active cell secretion through the exosomal pathway. Further, quantitative analysis of secreted infectivity indicated that the proportion of prions eventually secreted was dramatically dependent on the prion strain. Our data also highlight that infectious exosomes secreted from cultured cells might represent a biologically pertinent material for spiking experiments. Also discussed is the appealing possibility that abnormal PrP from different prion strains may differentially interact with the cellular machinery to promote secretion. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Adj. k-factor
256 (pelleting) / 256 (washing)
Protein markers
EV: Alix/ Flotilin1/ EF1A
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.17-1.2
Show all info
Study aim
Function
Sample
Species
Oryctolagus cuniculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
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: time(min)
75
Pelleting: rotor type
SW32
Pelleting: adjusted k-factor
256.0
Wash: Rotor Type
SW32
Wash: adjusted k-factor
256.0
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Rotor type
SW32
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Flotilin1/ EF1A
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
EF1A
Characterization: Particle analysis
EV140120 6/7 Homo sapiens Urine 0.2 µm filter
Other method
Ghosh A 2014 50%

Study summary

Full title
All authors
Ghosh A, Davey M, Chute IC, Griffiths SG, Lewis S, Chacko S, Barnett D, Crapoulet N, Fournier S, Joy A, Caissie MC, Ferguson AD, Daigle M, Meli MV, Lewis SM, Ouellette RJ
Journal
PLoS One
Abstract
Recent studies indicate that extracellular vesicles are an important source material for many clinic (show more...)Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications. (hide)
EV-METRIC
50% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Other method
Protein markers
EV: Alix/ CD63/ CD9/ HSP70/ CD24/ PSMA
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Other
Name other isolation method
Vn peptides
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD9/ HSP70/ CD24/ PSMA
ELISA
Detected EV-associated proteins
CD24/ PSMA
Characterization: Particle analysis
NTA
EM
EM-type
atomic force EM
Image type
Close-up, Wide-field
EV140016 2/2 Homo sapiens Saliva Commercial Zlotogorski-Hurvitz A 2014 50%

Study summary

Full title
All authors
Zlotogorski-Hurvitz A, Dayan D, Chaushu G, Korvala J, Salo T, Sormunen R, Vered M
Journal
J Histochem Cytochem
Abstract
ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for us (show more...)ExoQuick-TC(TM) (EQ), a chemical-based agent designed to precipitate exosomes, was calibrated for use on saliva collected from healthy individuals. The morphological and molecular features of the precipitations were compared with those obtained using the classical, physical-based method of ultracentrifugation (UC). Electron microscopy and immunoelectron microscopy with anti-CD63 showed vesicular nanoparticles surrounded by bi-layered membrane, compatible with exosomes in EQ, similar to that observed with UC. Atomic force microscopy highlighted larger, irregularly shaped/aggregated EQ nanoparticles that contrasted with the single, round-shaped UC nanoparticles. ELISA (performed on 0.5 ml of saliva) revealed a tendency for a higher expression of the specific exosomal markers (CD63, CD9, CD81) in EQ than in UC (p>0.05). ELISA for epithelial growth factor receptor, a non-exosomal-related marker, showed a significantly higher concentration in EQ than in UC (p=0.04). Western blotting of equal total-protein concentrations revealed bands of CD63, CD9 and CD81 in both types of preparations, although they were less pronounced in EQ compared with UC. This may be related to a higher fraction of non-exosomal proteins in EQ. In conclusion, EQ is suitable and efficient for precipitation of salivary exosomes from small volumes of saliva; however, EQ tends to be associated with considerably more biological impurities (non-exosomal-related proteins/microvesicles) as compared with UC. (hide)
EV-METRIC
50% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Saliva
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial
Protein markers
EV: CD63/ CD81/ CD9/ Beta-actin
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Saliva
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ Beta-actin
ELISA
Detected EV-associated proteins
CD63/ CD81/ CD9/ Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM/ atomic force EM
Image type
Close-up, Wide-field
EV140044 1/2 Rattus norvegicus/rattus Cell culture supernatant Commercial
UF
Wang X 2014 50%

Study summary

Full title
All authors
Wang X, Huang W, Liu G, Cai W, Millard RW, Wang Y, Chang J, Peng T, Fan GC
Journal
J Mol Cell Cardiol
Abstract
Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play crit (show more...)Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes. (hide)
EV-METRIC
50% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial + UF
Protein markers
EV: CD63/ CD81/ AChE
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ AChE
ELISA
Detected EV-associated proteins
AChE
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140015 1/1 Homo sapiens
Mus musculus
Cell culture supernatant 0.2 µm filter
Commercial
UF
Wang J 2014 50%

Study summary

Full title
All authors
Wang J, Hendrix A, Hernot S, Lemaire M, De Bruyne E, Van Valckenborgh E, Lahoutte T, De Wever O, Vanderkerken K, Menu E
Journal
Blood
Abstract
The interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells performs a c (show more...)The interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells performs a crucial role in MM pathogenesis by secreting growth factors, cytokines, and extracellular vesicles. Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and they mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. Although BMSC-induced growth and drug resistance of MM cells has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here we investigate the effect of BMSC-derived exosomes on the viability, proliferation, survival, migration, and drug resistance of MM cells, using the murine 5T33MM model and human MM samples. BMSCs and MM cells could mutually exchange exosomes carrying certain cytokines. Both naive and 5T33 BMSC-derived exosomes increased MM cell growth and induced drug resistance to bortezomib. BMSC-derived exosomes also influenced the activation of several survival relevant pathways, including c-Jun N-terminal kinase, p38, p53, and Akt. Exosomes obtained from normal donor and MM patient BMSCs also induced survival and drug resistance of human MM cells. Taken together, our results demonstrate the involvement of exosome-mediated communication in BMSC-induced proliferation, migration, survival, and drug resistance of MM cells. (hide)
EV-METRIC
50% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Commercial + UF
Protein markers
EV: CD63/ Flotilin1/ HSP90/ HSP70
non-EV: Cell organelle protein
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Homo sapiens / Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ HSP90/ HSP70
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140014 1/1 Homo sapiens Semen 0.2 µm filter
0.45 µm filter
Density cushion
UF
Vojtech L 2014 50%

Study summary

Full title
All authors
Vojtech L, Woo S, Hughes S, Levy C, Ballweber L, Sauteraud RP, Strobl J, Westerberg K, Gottardo R, Tewari M, Hladik F
Journal
Nucleic Acids Res
Abstract
Semen contains relatively ill-defined regulatory components that likely aid fertilization, but which (show more...)Semen contains relatively ill-defined regulatory components that likely aid fertilization, but which could also interfere with defense against infection. Each ejaculate contains trillions of exosomes, membrane-enclosed subcellular microvesicles, which have immunosuppressive effects on cells important in the genital mucosa. Exosomes in general are believed to mediate inter-cellular communication, possibly by transferring small RNA molecules. We found that seminal exosome (SE) preparations contain a substantial amount of RNA from 20 to 100 nucleotides (nts) in length. We sequenced 20-40 and 40-100 nt fractions of SE RNA separately from six semen donors. We found various classes of small non-coding RNA, including microRNA (21.7% of the RNA in the 20-40 nt fraction) as well as abundant Y RNAs and tRNAs present in both fractions. Specific RNAs were consistently present in all donors. For example, 10 (of ?2600 known) microRNAs constituted over 40% of mature microRNA in SE. Additionally, tRNA fragments were strongly enriched for 5'-ends of 18-19 or 30-34 nts in length; such tRNA fragments repress translation. Thus, SE could potentially deliver regulatory signals to the recipient mucosa via transfer of small RNA molecules. (hide)
EV-METRIC
50% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Semen
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + 0.45 µm filter + Density cushion + UF
Protein markers
EV: CD63/ HSP70
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Semen
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV140043 2/3 Homo sapiens Serum 0.45 µm filter
Commercial
Vargas A 2014 50%

Study summary

Full title
All authors
Vargas A, Zhou S, Éthier-Chiasson M, Flipo D, Lafond J, Gilbert C, Barbeau B
Journal
FASEB J
Abstract
Exosomes are extracellular vesicles that mediate intercellular communication and are involved in sev (show more...)Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women. (hide)
EV-METRIC
50% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + Commercial
Protein markers
EV: CD63/ TSG101/ AChE
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Isolation Method
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ AChE
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
CD63/ TSG101/ AChE
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Wide-field
EV140043 3/3 Homo sapiens Cell culture supernatant 0.45 µm filter
Commercial
UF
Vargas A 2014 50%

Study summary

Full title
All authors
Vargas A, Zhou S, Éthier-Chiasson M, Flipo D, Lafond J, Gilbert C, Barbeau B
Journal
FASEB J
Abstract
Exosomes are extracellular vesicles that mediate intercellular communication and are involved in sev (show more...)Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women. (hide)
EV-METRIC
50% (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
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + Commercial + UF
Protein markers
EV: CD63/ TSG101/ AChE
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ AChE
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
CD63/ TSG101/ AChE
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Wide-field
EV140132 3/4 Mus musculus Blood plasma Sucrose-DG (valid.) Povero D 2014 50%

Study summary

Full title
All authors
Povero D, Eguchi A, Li H, Johnson CD, Papouchado BG, Wree A, Messer K, Feldstein AE
Journal
PLoS One
Abstract
BACKGROUND & AIM: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease (show more...)BACKGROUND & AIM: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. DESIGN: Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. RESULTS: We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver. CONCLUSIONS: These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD. (hide)
EV-METRIC
50% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient