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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.
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  • 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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Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV130101 1/2 Homo sapiens Cell culture supernatant 0.1 µm filter
0.2 µm
dUC
Marimpietri D 2013 38%

Study summary

Full title
All authors
Marimpietri D, Petretto A, Raffaghello L, Pezzolo A, Gagliani C, Tacchetti C, Mauri P, Melioli G, Pistoia V
Journal
PLoS One
Abstract
Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, with grim prognosis in (show more...)Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs) of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133), basigin (CD147) and B7-H3 (CD276). Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers. (hide)
EV-METRIC
38% (81st 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
0.1 µm filter + 0.2 µm + dUC
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: CD63/ CD81/ CD9/ Fibronectin
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
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)
75
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
156.9
Wash: Rotor Type
70Ti
Wash: adjusted k-factor
156.9
Filtration steps
0.22µm or 0.2µm0.2µm > x > 0.1µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Fibronectin
ELISA
Detected EV-associated proteins
Fibronectin
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Wide-field
EV130100 1/2 Rattus norvegicus/rattus Cell culture supernatant Commercial
UF
Malik ZA 2013 38%

Study summary

Full title
All authors
Malik ZA, Kott KS, Poe AJ, Kuo T, Chen L, Ferrara KW, Knowlton AA
Journal
Am J Physiol Heart Circ Physiol
Abstract
Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular com (show more...)Exosomes, which are 50- to 100-nm-diameter lipid vesicles, have been implicated in intercellular communication, including transmitting malignancy, and as a way for viral particles to evade detection while spreading to new cells. Previously, we demonstrated that adult cardiac myocytes release heat shock protein (HSP)60 in exosomes. Extracellular HSP60, when not in exosomes, causes cardiac myocyte apoptosis via the activation of Toll-like receptor 4. Thus, release of HSP60 from exosomes would be damaging to the surrounding cardiac myocytes. We hypothesized that 1) pathological changes in the environment, such as fever, change in pH, or ethanol consumption, would increase exosome permeability; 2) different exosome inducers would result in different exosomal protein content; 3) ethanol at physiological concentrations would cause exosome release; and 4) ROS production is an underlying mechanism of increased exosome production. We found the following: first, exosomes retained their protein cargo under different physiological/pathological conditions, based on Western blot analyses. Second, mass spectrometry demonstrated that the protein content of cardiac exosomes differed significantly from other types of exosomes in the literature and contained cytosolic, sarcomeric, and mitochondrial proteins. Third, ethanol did not affect exosome stability but greatly increased the production of exosomes by cardiac myocytes. Fourth, ethanol- and hypoxia/reoxygenation-derived exosomes had different protein content. Finally, ROS inhibition reduced exosome production but did not completely inhibit it. In conclusion, exosomal protein content is influenced by the cell source and stimulus for exosome formation. ROS stimulate exosome production. The functions of exosomes remain to be fully elucidated. (hide)
EV-METRIC
38% (81st 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: HSP60/ GAPDH/ AChE
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
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
HSP60/ GAPDH/ AChE
ELISA
Detected EV-associated proteins
HSP60/ GAPDH/ AChE
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV130040 1/1 Homo sapiens Blood plasma Other-DG Gourzones C 2013 38%

Study summary

Full title
All authors
Gourzones C, Ferrand FR, Amiel C, Vérillaud B, Barat A, Guérin M, Gattolliat CH, Gelin A, Klibi J, Chaaben AB, Schneider V, Guemira F, Guigay J, Lang P, Jimenez-Pailhes AS, Busson P
Journal
Virol J
Abstract
BACKGROUND: Because latent Epstein Barr (EBV)-infection is a specific characteristic of malignant na (show more...)BACKGROUND: Because latent Epstein Barr (EBV)-infection is a specific characteristic of malignant nasopharyngeal carcinoma (NPC), various molecules of viral origin are obvious candidate biomarkers in this disease. In a previous study, we could show in a few clinical samples that it was possible to detect a category of EBV microRNAs called miR-BARTs in the plasma of at least a fraction of NPC patients. The first aim of the present study was to investigate the status of circulating miR-BART17-5p (one of the miR-BARTs hereafter called miR-BART17) and EBV DNA in a larger series of NPC plasma samples. The second aim was to determine whether or not circulating miR-BART17 was carried by plasma exosomes. PATIENTS AND METHODS: Plasma samples were collected from 26 NPC patients and 10 control donors, including 9 patients with non-NPC Head and Neck squamous cell carcinoma and one healthy EBV carrier. Concentrations of miR-BART17 and two cellular microRNAs (hsa-miR-16 and -146a) were assessed by real-time quantitative PCR with spike-in normalization and absolute quantification. In addition, for 2 patients, exosome distributions of miR-BART17 and miR-16 were investigated following plasma lipoprotein fractionation by isopycnic density gradient ultrcentrifugation. RESULTS: The miR-BART17 was significantly more abundant in plasma samples from NPC patients compared to non-NPC donors. Above a threshold of 506 copies/mL, detection of miR-BART17 was highly specific for NPC patients (ROC curve analysis: AUC=0.87 with true positive rate = 0.77, false positive rate = 0.10). In this relatively small series, the concentration of plasma miR-BART17 and the plasma EBV DNA load were not correlated. When plasma samples were fractionated, miR-BART17 co-purified with a protein-rich fraction but not with exosomes. CONCLUSIONS: Detection of high concentrations of plasma miR-BART17 is consistent in NPC patients. This parameter is, at least in part, independent of the viral DNA load. Circulating miR-BART17 does not co-purify with exosomes. (hide)
EV-METRIC
38% (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
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
Other-DG
Protein markers
EV: CD63
non-EV: None
Proteomics
no
EV density (g/ml)
1.15-1.19
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Density gradient
Lowest density fraction
1.006g/L
Highest density fraction
1.24g/L
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63
Characterization: Particle analysis
EV130026 2/3 Homo sapiens Urine UF Gerlach JQ 2013 38%

Study summary

Full title
All authors
Gerlach JQ, Krüger A, Gallogly S, Hanley SA, Hogan MC, Ward CJ, Joshi L, Griffin MD
Journal
PLoS One
Abstract
Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomo (show more...)Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications. (hide)
EV-METRIC
38% (79th 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
UF
Protein markers
EV: CD24/ AQP2
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 800 g and 10,000 g
Between 10,000 g and 50,000 g
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD24/ AQP2
ELISA
Detected EV-associated proteins
CD24/ AQP2
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
EV130034 1/1 Homo sapiens Semen Density cushion
SEC
Sucrose-DG
Brouwers JF 2013 38%

Study summary

Full title
All authors
Brouwers JF, Aalberts M, Jansen JW, van Niel G, Wauben MH, Stout TA, Helms JB, Stoorvogel W
Journal
Proteomics
Abstract
Prostasomes are vesicles secreted by prostate epithelial cells and found in abundance in seminal pla (show more...)Prostasomes are vesicles secreted by prostate epithelial cells and found in abundance in seminal plasma. They regulate aspects of sperm cell function and are also thought to prevent immune-mediated destruction of sperm cells within the female reproductive tract. In a previous study, we isolated two distinct populations of prostasomes, differing both in size and protein composition, from the seminal fluid of vasectomized men. In the current study, we characterized the lipid content of these two prostasome populations. Both prostasome types had an unusual lipid composition, with high levels of sphingomyelin (SM), cholesterol, and glycosphingolipids at the expense of, in particular, phosphatidylcholine. The different classes of glycerophospholipids consisted mainly of mono-unsaturated species. The sphingosine-based lipids, SM and the hexosylceramides, were characterized by a near absence of unsaturated species. The two types of prostasome differed in lipid composition, particularly with regard to the relative contributions of SM and hexosylceramides. Potential implications of the lipid compositions of prostasomes for the mechanisms of their formation and function are discussed. (hide)
EV-METRIC
38% (69th 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
Prostasomes
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
Density cushion + SEC + Sucrose-DG
Protein markers
EV: CD9
non-EV: PSCA
Proteomics
no
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Semen
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Density gradient
Lowest density fraction
0.4
Highest density fraction
2
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9
Detected contaminants
PSCA
Characterization: Particle analysis
EM
EM-type
cryo EM
Image type
Close-up
EV130032 1/1 Homo sapiens Cell culture supernatant 0.1 µm filter
Iodixanol-DG
UF
Bernhard OK 2013 38%

Study summary

Full title
All authors
Bernhard OK, Greening DW, Barnes TW, Ji H, Simpson RJ
Journal
Biochim Biophys Acta Proteins Proteom
Abstract
Colorectal cancer (CRC), one of the most prevalent cancers in the western world, is treatable if det (show more...)Colorectal cancer (CRC), one of the most prevalent cancers in the western world, is treatable if detected early. However, 70% of CRC is detected at an advanced stage. This is largely due to the inadequacy of current faecal occult blood screening testing and costs involved in conducting population-based colonoscopy, the gold standard for CRC detection. Another biomarker for CRC, carcinoembryonic antigen, while useful for monitoring CRC recurrence, is ineffective, lacking the specificity required early detection of CRC. For these reasons there is a need for more effective blood-based markers for early CRC detection. In this study we targeted glycoproteins secreted from the human colon carcinoma cell line LIM1215 as a source of potential CRC biomarkers. Secreted candidate glycoproteins were confirmed by MS and validated by Western blot analysis of tissue/tumour interstitial fluid (Tif) from LIM1215 xenograft tumours grown in immunocompromised mice. Overall, 39 glycoproteins were identified in LIM1215 culture media (CCM) and 5 glycoproteins in LIM1215 tumour xenograft Tif; of these, cadherin-17 (CDH17), galectin-3 binding protein (LGALS3BP), and tyrosine-protein kinase-like 7 (PTK7) were identified in both CM and glycosylation motifs. Swiss-Prot was used to annotate Tif. Many of the glycoproteins identified in this study (e.g., AREG, DSG2, EFNA1, EFNA3, EFNA4, EPHB4, ST14, and TIMP1) have been reported to be implicated in CRC biology. Interestingly, the cadherin-17 ectodomain, but not full length cadherin-17, was identified in CM, Tif and plasma derived from mice bearing the LIM1215 xenograft tumour. To our knowledge, this is the first report of the cadherin-17 ectodomain in plasma. In this study, we report for the first time that the presence of full-length cadherin-17 in exosomes released into the CM. This article is part of a Special Issue entitled: An Updated Secretome. (hide)
EV-METRIC
38% (81st 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
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 + Iodixanol-DG + UF
Protein markers
EV: CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.110
Show all info
Study aim
Biomarker
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
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Speed (g)
100000
Filtration steps
0.2µm > x > 0.1µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9
Characterization: Particle analysis
EV130135 2/2 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
Lee JH 2013 33%

Study summary

Full title
All authors
Lee JH, Wittki S, Bräu T, Dreyer FS, Krätzel K, Dindorf J, Johnston IC, Gross S, Kremmer E, Zeidler R, Schlötzer-Schrehardt U, Lichtenheld M, Saksela K, Harrer T, Schuler G, Federico M, Baur AS
Journal
Mol Cell
Abstract
The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for rea (show more...)The HIV Nef protein recruits the polycomb protein Eed and mimics an integrin receptor signal for reasons that are not entirely clear. Here we demonstrate that Nef and Eed complex with the integrin effector paxillin to recruit and activate TNF? converting enzyme (TACE alias ADAM 17) and its close relative ADAM10. The activated proteases cleaved proTNF? and were shuttled into extracellular vesicles (EVs). Peripheral blood mononuclear cells that ingested these EVs released TNF?. Analyzing the mechanism, we found that Pak2, an established host cell effector of Nef, phosphorylated paxillin on Ser272/274 to induce TACE-paxillin association and shuttling into EVs via lipid rafts. Conversely, Pak1 phosphorylated paxillin on Ser258, which inhibited TACE association and lipid raft transfer. Interestingly, melanoma cells used an identical mechanism to shuttle predominantly ADAM10 into EVs. We conclude that HIV-1 and cancer cells exploit a paxillin/integrin-controlled mechanism to release TACE/ADAM10-containing vesicles, ensuring better proliferation/growth conditions in their microenvironment. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
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: CD63/ CD81/ CD9/ MHC1/ ADAM17
non-EV: None
Proteomics
no
EV density (g/ml)
1.12-1.23
Show all info
Study aim
Other/Regulation of ADAM10 activation and secretion
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
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
Wash: volume per pellet (ml)
35
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Speed (g)
110000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ MHC1/ ADAM17
ELISA
Detected EV-associated proteins
MHC1/ ADAM17
Characterization: Particle analysis
EM
EM-type
transmission EM/ scanning EM
Image type
Wide-field
EV130027 1/4 Mus musculus Cell culture supernatant dUC Hajj GN 2013 33%

Study summary

Full title
All authors
Hajj GN, Arantes CP, Dias MV, Roffé M, Costa-Silva B, Lopes MH, Porto-Carreiro I, Rabachini T, Lima FR, Beraldo FH, Prado MA, Linden R, Martins VR
Journal
Cell Mol Life Sci
Abstract
The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neur (show more...)The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36
non-EV: "GAPDH/ LAMP1"
Proteomics
no
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
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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36
Detected contaminants
"GAPDH/ LAMP1"
ELISA
Detected EV-associated proteins
PrP/ Tf-receptor/ VPS36
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV130027 2/4 Mus musculus Cell culture supernatant dUC Hajj GN 2013 33%

Study summary

Full title
All authors
Hajj GN, Arantes CP, Dias MV, Roffé M, Costa-Silva B, Lopes MH, Porto-Carreiro I, Rabachini T, Lima FR, Beraldo FH, Prado MA, Linden R, Martins VR
Journal
Cell Mol Life Sci
Abstract
The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neur (show more...)The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36
non-EV: "GAPDH/ LAMP1"
Proteomics
no
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
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)
960
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36
Detected contaminants
"GAPDH/ LAMP1"
ELISA
Detected EV-associated proteins
PrP/ Tf-receptor/ VPS36
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV130027 3/4 Mus musculus Cell culture supernatant dUC Hajj GN 2013 33%

Study summary

Full title
All authors
Hajj GN, Arantes CP, Dias MV, Roffé M, Costa-Silva B, Lopes MH, Porto-Carreiro I, Rabachini T, Lima FR, Beraldo FH, Prado MA, Linden R, Martins VR
Journal
Cell Mol Life Sci
Abstract
The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neur (show more...)The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36
non-EV: "GAPDH/ LAMP1"
Proteomics
no
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
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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP90/ HSP70/ TSG101/ PrP/ Tf-receptor/ VPS36
Detected contaminants
"GAPDH/ LAMP1"
ELISA
Detected EV-associated proteins
PrP/ Tf-receptor/ VPS36
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV130072 2/4 Homo sapiens Cell culture supernatant dUC
Sucrose-DG
Zeringer E 2013 33%

Study summary

Full title
All authors
Zeringer E, Li M, Barta T, Schageman J, Pedersen KW, Neurauter A, Magdaleno S, Setterquist R, Vlassov AV
Journal
World J Methodol
Abstract
AIM: To develop protocols for isolation of exosomes and characterization of their RNA content. METHO (show more...)AIM: To develop protocols for isolation of exosomes and characterization of their RNA content. METHODS: Exosomes were extracted from HeLa cell culture media and human blood serum using the Total exosome isolation (from cell culture media) reagent, and Total exosome isolation (from serum) reagent respectively. Identity and purity of the exosomes was confirmed by Nanosight(®) analysis, electron microscopy, and Western blots for CD63 marker. Exosomal RNA cargo was recovered with the Total exosome RNA and protein isolation kit. Finally, RNA was profiled using Bioanalyzer and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) methodology. RESULTS: Here we describe a novel approach for robust and scalable isolation of exosomes from cell culture media and serum, with subsequent isolation and analysis of RNA residing within these vesicles. The isolation procedure is completed in a fraction of the time, compared to the current standard protocols utilizing ultracentrifugation, and allows to recover fully intact exosomes in higher yields. Exosomes were found to contain a very diverse RNA cargo, primarily short sequences 20-200 nt (such as miRNA and fragments of mRNA), however longer RNA species were detected as well, including full-length 18S and 28S rRNA. CONCLUSION: We have successfully developed a set of reagents and a workflow allowing fast and efficient extraction of exosomes, followed by isolation of RNA and its analysis by qRT-PCR and other techniques. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
non-EV: None
Proteomics
no
EV density (g/ml)
1.13-1.19
Show all info
Study aim
Technical
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Speed (g)
110000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63
Characterization: Particle analysis
NTA
EV130071 2/2 Bos bovis Milk 0.2 µm filter
0.45 µm
dUC
Sucrose-DG (valid.)
Yamada T 2013 33%

Study summary

Full title
All authors
Yamada T, Shigemura H, Ishiguro N, Inoshima Y
Journal
PLoS One
Abstract
Exosomes are small membranous microvesicles (40-100 nm in diameter) and are extracellularly released (show more...)Exosomes are small membranous microvesicles (40-100 nm in diameter) and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV) infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exosomes and BLV in bovine milk. BLV structural proteins, gp51 (Env) and p24 (Gag), were detected in bovine milk exosomes from BLV-infected cattle by Western blot analysis. In cells inoculated with these milk exosomes, BLV DNA was not detected during three serial passages by nested PCR. Purification of exosomes from persistently BLV-infected cells was achieved by immuno-magnetic separation using an antibody against exosomes coupled to magnetic beads. Consistently, BLV gp51 and p24 proteins were detected in purified exosomes. Moreover, reverse transcriptase activity was observed in purified exosomes, meaning that exosomes also contain viral enzyme. However, BLV DNA was not detected in serially passaged cells after inoculation of purified exosomes, indicating that exosomes carrying BLV proteins appeared to be not infectious. These results suggest that BLV proteins are released with milk exosomes and could be transferred into recipient cells of calves via milk exosomes as an alternative route not requiring virus infection. Moreover it is also possible that bovine milk exosomes play a role in clearance of BLV proteins from infected cells. (hide)
EV-METRIC
33% (62nd 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
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 + dUC + Sucrose-DG (valid.)
Protein markers
EV: MFGE8
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.19
Show all info
Study aim
Omics
Sample
Species
Bos bovis
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
Between 100,000 g and 150,000 g
Between 50,000 g and 100,000 g
Pelleting: time(min)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
MFGE8
ELISA
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
EV130059 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
van Balkom BW 2013 33%

Study summary

Full title
All authors
van Balkom BW, de Jong OG, Smits M, Brummelman J, den Ouden K, de Bree PM, van Eijndhoven MA, Pegtel DM, Stoorvogel W, Würdinger T, Verhaar MC
Journal
Blood
Abstract
Signaling between endothelial cells, endothelial progenitor cells, and stromal cells is crucial for (show more...)Signaling between endothelial cells, endothelial progenitor cells, and stromal cells is crucial for the establishment and maintenance of vascular integrity and involves exosomes, among other signaling pathways. Exosomes are important mediators of intercellular communication in immune signaling, tumor survival, stress responses, and angiogenesis. The ability of exosomes to incorporate and transfer messenger RNAs (mRNAs) encoding for acquired proteins or micro RNAs (miRNAs) repressing resident mRNA translation suggests that they can influence the physiological behavior of recipient cells. We demonstrate that miR-214, an miRNA that controls endothelial cell function and angiogenesis, plays a dominant role in exosome-mediated signaling between endothelial cells. Endothelial cell-derived exosomes stimulated migration and angiogenesis in recipient cells, whereas exosomes from miR-214-depleted endothelial cells failed to stimulate these processes. Exosomes containing miR-214 repressed the expression of ataxia telangiectasia mutated in recipient cells, thereby preventing senescence and allowing blood vessel formation. Concordantly, specific reduction of miR-214 content in exosome-producing endothelial cells abolishes the angiogenesis stimulatory function of the resulting exosomes. Collectively, our data indicate that endothelial cells release miR-214-containing exosomes to stimulate angiogenesis through the silencing of ataxia telangiectasia mutated in neighboring target cells. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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.)
Protein markers
EV: Flotilin1
non-EV: None
Proteomics
no
EV density (g/ml)
1.110
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)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV130121 1/2 Homo sapiens Cell culture supernatant Sucrose-DG Tan SS 2013 33%

Study summary

Full title
All authors
Tan SS, Yin Y, Lee T, Lai RC, Yeo RW, Zhang B, Choo A, Lim SK
Journal
J Extracell Vesicles
Abstract
BACKGROUND: Mesenchymal stem cell (MSC) was previously shown to secrete lipid vesicles that when pur (show more...)BACKGROUND: Mesenchymal stem cell (MSC) was previously shown to secrete lipid vesicles that when purified by high performance liquid chromatography as a population of homogenously sized particles with a hydrodynamic radius of 55-65 nm reduce infarct size in a mouse model of myocardial ischemia/reperfusion injury. As these vesicles exhibit many biophysical and biochemical properties of exosomes, they were identified as exosomes. Here we investigated if these lipid vesicles were indeed exosomes that have an endosomal biogenesis. METHOD: In most cells, endocytosis is thought to occur at specialized microdomains known as lipid rafts. To demonstrate an endosomal origin for MSC exosomes, MSCs were pulsed with ligands e.g. transferrin (Tfs) and Cholera Toxin B (CTB) that bind receptors in lipid rafts. The endocytosed ligands were then chased to determine if they were incorporated into the exosomes. RESULTS: A fraction of exogenous Tfs was found to recycle into MSC exosomes. When MSCs were pulsed with labelled Tfs in the presence of chlorpromazine, an inhibitor of clathrin-mediated endocytosis, Tf incorporation in CD81-immunoprecipitate was reduced during the chase. CTB which binds GM1 gangliosides that are enriched in lipid rafts extracted exosome-associated proteins, CD81, CD9, Alix and Tsg101 from MSC-conditioned medium. Exogenous CTBs were pulse-chased into secreted vesicles. Extraction of Tf- or CTB-binding vesicles in an exosome preparation mutually depleted each other. Inhibition of sphingomyelinases reduced CTB-binding vesicles. CONCLUSION: Together, our data demonstrated that MSC exosomes are derived from endocytosed lipid rafts and that their protein cargo includes exosome-associated proteins CD81, CD9, Alix and Tsg101. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Sucrose-DG
Protein markers
EV: CD81
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.17
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Density gradient
Lowest density fraction
22.8
Highest density fraction
60
Orientation
Top-down
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EV130118 1/1 Homo sapiens Blood plasma 0.2 µm filter
dUC
Szajnik M 2013 33%

Study summary

Full title
All authors
Szajnik M, Derbis M, Lach M, Patalas P, Michalak M, Drzewiecka H, Szpurek D, Nowakowski A, Spaczynski M, Baranowski W, Whiteside TL
Journal
Gynecol Obstet (Sunnyvale)
Abstract
BACKGROUND: In patients with Ovarian Cancer (OvCa) exosomes released by tumor cells are present in t (show more...)BACKGROUND: In patients with Ovarian Cancer (OvCa) exosomes released by tumor cells are present in the plasma and could be involved in tumor progression. This study examines the association between the exosome presence/protein content in plasma of OvCa patients and disease outcome, response to standard therapy and/or tumorresistance to therapies in patients studied at diagnosis and also serially during and after therapy. DESIGN AND METHODS: Exosomes were purified from OvCa patients' plasma (n=22), patients with benign tumors (n=10) or (n=10) healthy controls (NC) using ultracentrifugation. Exosomes were visualized by scanning electron microscopy. Their protein content was measured. The presence of MAGE 3/6 and TGF-?1 in exosomes was evaluated in Western blots. RESULTS: The OvCa patients' plasma contained higher levels of exosomal proteins (p<0.05) compared to those isolated from plasma of patients with benign tumors or NC. Exosomes isolated from OvCa patients's plasma carried TGF-?1 and MAGE3/6, which distinguished OvCa patients from those with benign tumors and NC. High protein levels of exosomes were seen in newly diagnosed patients; however in advanced stages of OvCa patients the protein content of isolated exosomes was significantly higher than that of early stages. The exosome levels variably changed during/after chemotherapy, and correlations between the changes in exosomal protein levels and clinical data suggested that the protein content of exosomes might be useful in predicting responses to therapy and prognosis in OvCa patients. CONCLUSION: Analysis of plasma exosomes levels offers a novel approach to diagnosis and monitoring response to therapies in OvCa patients. (hide)
EV-METRIC
33% (79th 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
Adj. k-factor
101.8 (pelleting)
Protein markers
EV: LAMP1
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
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)
90
Pelleting: rotor type
70.1Ti
Pelleting: adjusted k-factor
101.8
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
LAMP1
ELISA
Detected EV-associated proteins
LAMP1
Characterization: Particle analysis
EM
EM-type
scanning EM
Image type
Wide-field
EV130019 1/1 Homo sapiens Cell culture supernatant dUC Svensson KJ 2013 33%

Study summary

Full title
All authors
Svensson KJ, Christianson HC, Wittrup A, Bourseau-Guilmain E, Lindqvist E, Svensson LM, Mörgelin M, Belting M
Journal
J Biol Chem
Abstract
The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell de (show more...)The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: CD63/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ immune EM
Image type
Close-up
EV130057 1/1 Homo sapiens Placental explant 0.2 µm filter
dUC
Sucrose-DG
Stenqvist AC 2013 33%

Study summary

Full title
All authors
Stenqvist AC, Nagaeva O, Baranov V, Mincheva-Nilsson L
Journal
J Immunol
Abstract
Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We inv (show more...)Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We investigated the expression and in vitro apoptotic activity of two physiologically relevant death messengers, the TNF family members Fas ligand (FasL) and TRAIL in human early and term placentas. Both molecules were intracellularly expressed, confined to the late endosomal compartment of the syncytiotrophoblast, and tightly associated to the generation and secretion of placental exosomes. Using immunoelectron microscopy, we show that FasL and TRAIL are expressed on the limiting membrane of multivesicular bodies where, by membrane invagination, intraluminal microvesicles carrying membranal bioactive FasL and TRAIL are formed and released in the extracellular space as exosomes. Analyzing exosomes secreted from placental explant cultures, to our knowledge, we demonstrate for the first time that FasL and TRAIL are clustered on the exosomal membrane as oligomerized aggregates ready to form death-inducing signaling complex. Consistently, placental FasL- and TRAIL-carrying exosomes triggered apoptosis in Jurkat T cells and activated PBMC in a dose-dependent manner. Limiting the expression of functional FasL and TRAIL to exosomes comprise a dual benefit: 1) storage of exosomal FasL and TRAIL in multivesicular bodies is protected from proteolytic cleavage and 2) upon secretion, delivery of preformed membranal death molecules by exosomes rapidly triggers apoptosis. Our results suggest that bioactive FasL- and TRAIL-carrying exosomes, able to convey apoptosis, are secreted by the placenta and tie up the immunomodulatory and protective role of human placenta to its exosome-secreting ability. (hide)
EV-METRIC
33% (62nd 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
Placental explant
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
Protein markers
EV: CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Placental explant
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
20
Highest density fraction
40
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV130056 1/2 Bos bovis Follicular fluid 0.2 µm filter
dUC
Sohel MM 2013 33%

Study summary

Full title
All authors
Sohel MM, Hoelker M, Noferesti SS, Salilew-Wondim D, Tholen E, Looft C, Rings F, Uddin MJ, Spencer TE, Schellander K, Tesfaye D
Journal
PLoS One
Abstract
Cell-cell communication within the follicle involves many signaling molecules, and this process may (show more...)Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. (hide)
EV-METRIC
33% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
Follicular fluid
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
138.6 (pelleting) / 138.6 (washing)
Protein markers
EV: CD63
non-EV: Cell organelle protein/ Ago2
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Bos bovis
Sample Type
Follicular fluid
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
SW55
Pelleting: adjusted k-factor
138.6
Wash: Rotor Type
SW55
Wash: adjusted k-factor
138.6
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63
Detected contaminants
Cell organelle protein/ Ago2
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV130114 1/3 Homo sapiens Cell culture supernatant dUC Shin SJ 2013 33%

Study summary

Full title
All authors
Shin SJ, Smith JA, Rezniczek GA, Pan S, Chen R, Brentnall TA, Wiche G, Kelly KA
Journal
Proc Natl Acad Sci U S A
Abstract
We recently demonstrated that plectin is a robust biomarker for pancreatic ductal adenocarcinoma (PD (show more...)We recently demonstrated that plectin is a robust biomarker for pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive malignancies. In normal physiology, plectin is an intracellular scaffolding protein, but we have demonstrated localization on the extracellular surface of PDAC cells. In this study, we confirmed cell surface localization. Interestingly, we found that plectin cell surface localization was attributable to its presence in exosomes secreted from PDAC cells, which is dependent on the expression of integrin ?4, a protein known to interact with cytosolic plectin. Moreover, plectin expression was necessary for efficient exosome production and was required to sustain enhanced tumor growth in immunodeficient and in immunocompetent mice. It is now clear that this PDAC biomarker plays a role in PDAC, and further understanding of plectin's contribution to PDAC could enable improved therapies. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
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
104.8 (pelleting)
Protein markers
EV: Alix/ CD63/ CD81/ CD9/ HSP90/ HSP70
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
960
Pelleting: rotor type
45Ti
Pelleting: adjusted k-factor
104.8
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD81/ CD9/ HSP90/ HSP70
Characterization: Particle analysis
DLS
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV130055 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
Density cushion
dUC
Salomon C 2013 33%

Study summary

Full title
All authors
Salomon C, Ryan J, Sobrevia L, Kobayashi M, Ashman K, Mitchell M, Rice GE
Journal
PLoS One
Abstract
Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculatur (show more...)Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC) are known to release paracrine factors (some of which are contained within exosomes) that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC). pMSC were isolated from placental villi (8-12 weeks of gestation, n = 6) and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC) isolated by differential and buoyant density centrifugation. The dose effect (5-20 µg exosomal protein/ml) of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™). The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS). Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2) exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01). Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05) and increased hPMEC tube formation by 7.2 fold (p<0.05). MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both physiological and pathological conditions. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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 + Density cushion + dUC
Adj. k-factor
266.3 (pelleting)
Protein markers
EV: CD63/ CD81/ CD9
non-EV: None
Proteomics
yes
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 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
Surespin630/36
Pelleting: adjusted k-factor
266.3
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV130054 1/1 Homo sapiens Cell culture supernatant Iodixanol-DG (valid.) Regev-Rudzki N 2013 33%

Study summary

Full title
All authors
Regev-Rudzki N, Wilson DW, Carvalho TG, Sisquella X, Coleman BM, Rug M, Bursac D, Angrisano F, Gee M, Hill AF, Baum J, Cowman AF
Journal
Cell
Abstract
Cell-cell communication is an important mechanism for information exchange promoting cell survival f (show more...)Cell-cell communication is an important mechanism for information exchange promoting cell survival for the control of features such as population density and differentiation. We determined that Plasmodium falciparum-infected red blood cells directly communicate between parasites within a population using exosome-like vesicles that are capable of delivering genes. Importantly, communication via exosome-like vesicles promotes differentiation to sexual forms at a rate that suggests that signaling is involved. Furthermore, we have identified a P. falciparum protein, PfPTP2, that plays a key role in efficient communication. This study reveals a previously unidentified pathway of P. falciparum biology critical for survival in the host and transmission to mosquitoes. This identifies a pathway for the development of agents to block parasite transmission from the human host to the mosquito. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Iodixanol-DG (valid.)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Density gradient
Only used for validation of main results
1
Lowest density fraction
10
Highest density fraction
30
Orientation
Top-down
Characterization: Particle analysis
EM
EM-type
transmission EM/ cryo EM/ atomic force EM
Image type
Close-up, Wide-field
EV130053 1/2 Homo sapiens Cell culture supernatant dUC Rappa G 2013 33%

Study summary

Full title
All authors
Rappa G, Mercapide J, Anzanello F, Pope RM, Lorico A
Journal
Mol Cancer
Abstract
Exosomes can be viewed as complex messages packaged to survive trips to other cells in the local mic (show more...)Exosomes can be viewed as complex messages packaged to survive trips to other cells in the local microenvironment and, through body fluids, to distant sites. A large body of evidence indicates a pro-metastatic role for certain types of cancer exosomes. We previously reported that prominin-1 had a pro-metastatic role in melanoma cells and that microvesicles released from metastatic melanoma cells expressed high levels of prominin-1. With the goal to explore the mechanisms that govern proteo-lipidic-microRNA sorting in cancer exosomes and their potential contribution(s) to the metastatic phenotype, we here employed prominin-1-based immunomagnetic separation in combination with filtration and ultracentrifugation to purify prominin-1-expressing exosomes (prom1-exo) from melanoma and colon carcinoma cells. Prom1-exo contained 154 proteins, including all of the 14 proteins most frequently expressed in exosomes, and multiple pro-metastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10 and Annexin A2. Their lipid composition resembled that of raft microdomains, with a great enrichment in lyso-phosphatidylcholine, lyso-phosphatidyl-ethanolamine and sphingomyelin. The abundance of tetraspanins and of tetraspanin-associated proteins, together with the high levels of sphingomyelin, suggests that proteolipidic assemblies, probably tetraspanin webs, might be the essential structural determinant in the release process of prominin-1 of stem and cancer stem cells. Micro-RNA profiling revealed 49 species of micro-RNA present at higher concentrations in prom1-exo than in parental cells, including 20 with cancer-related function. Extensive accumulation of prom1-exo was observed 3 h after their addition to cultures of melanoma and bone marrow-derived stromal cells (MSC). Short-term co-culture of melanoma cells and MSC resulted in heterologous prominin-1 transfer. Exposure of MSC to prom1-exo increased their invasiveness. Our study supports the concept that specific populations of cancer exosomes contain multiple determinants of the metastatic potential of the cells from which they are derived. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: Alix
non-EV: None
Proteomics
yes
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
Equal to or above 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix
Characterization: Particle analysis
DLS
NTA
EV130053 2/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Immunoaffinity (valid.)
UF
Rappa G 2013 33%

Study summary

Full title
All authors
Rappa G, Mercapide J, Anzanello F, Pope RM, Lorico A
Journal
Mol Cancer
Abstract
Exosomes can be viewed as complex messages packaged to survive trips to other cells in the local mic (show more...)Exosomes can be viewed as complex messages packaged to survive trips to other cells in the local microenvironment and, through body fluids, to distant sites. A large body of evidence indicates a pro-metastatic role for certain types of cancer exosomes. We previously reported that prominin-1 had a pro-metastatic role in melanoma cells and that microvesicles released from metastatic melanoma cells expressed high levels of prominin-1. With the goal to explore the mechanisms that govern proteo-lipidic-microRNA sorting in cancer exosomes and their potential contribution(s) to the metastatic phenotype, we here employed prominin-1-based immunomagnetic separation in combination with filtration and ultracentrifugation to purify prominin-1-expressing exosomes (prom1-exo) from melanoma and colon carcinoma cells. Prom1-exo contained 154 proteins, including all of the 14 proteins most frequently expressed in exosomes, and multiple pro-metastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10 and Annexin A2. Their lipid composition resembled that of raft microdomains, with a great enrichment in lyso-phosphatidylcholine, lyso-phosphatidyl-ethanolamine and sphingomyelin. The abundance of tetraspanins and of tetraspanin-associated proteins, together with the high levels of sphingomyelin, suggests that proteolipidic assemblies, probably tetraspanin webs, might be the essential structural determinant in the release process of prominin-1 of stem and cancer stem cells. Micro-RNA profiling revealed 49 species of micro-RNA present at higher concentrations in prom1-exo than in parental cells, including 20 with cancer-related function. Extensive accumulation of prom1-exo was observed 3 h after their addition to cultures of melanoma and bone marrow-derived stromal cells (MSC). Short-term co-culture of melanoma cells and MSC resulted in heterologous prominin-1 transfer. Exposure of MSC to prom1-exo increased their invasiveness. Our study supports the concept that specific populations of cancer exosomes contain multiple determinants of the metastatic potential of the cells from which they are derived. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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 + Immunoaffinity (valid.) + UF
Protein markers
EV: Alix
non-EV: None
Proteomics
yes
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
Equal to or above 150,000 g
Pelleting: time(min)
60
Filtration steps
0.22µm or 0.2µm
Immunoaffinity capture
Selected surface protein(s)
prominin-1
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix
Characterization: Particle analysis
DLS
NTA
EV130153 2/3 Homo sapiens Urine dUC
Sucrose-DG (valid.)
Oosthuyzen W 2013 33%

Study summary

Full title
All authors
Oosthuyzen W, Sime NE, Ivy JR, Turtle EJ, Street JM, Pound J, Bath LE, Webb DJ, Gregory CD, Bailey MA, Dear JW
Journal
J Physiol
Abstract
Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA fro (show more...)Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi-quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24- and AQP2-positive particles of characteristic exosomal size. Extensive pre-NTA processing of urine was not necessary. However, the intra-assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at -80°C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine. (hide)
EV-METRIC
33% (72nd 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.)
Protein markers
EV: Flotilin1/ TSG101/ CD24
non-EV: None
Proteomics
no
EV density (g/ml)
1.12-1.16
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
Equal to or above 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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ TSG101/ CD24
ELISA
Detected EV-associated proteins
CD24
Characterization: Particle analysis
EV130065 3/3 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Kulshreshtha A 2013 33%

Study summary

Full title
All authors
Kulshreshtha A, Ahmad T, Agrawal A, Ghosh B
Journal
J Allergy Clin Immunol
Abstract
BACKGROUND: Exosomes are nanovesicles involved in intercellular communication. Their roles in variou (show more...)BACKGROUND: Exosomes are nanovesicles involved in intercellular communication. Their roles in various diseases are often contextual, depending on the cell type producing them. Although few studies hint toward the proinflammatory role of bronchoalveolar lavage fluid-derived exosomes in asthmatic progression, the cell types in lungs associated with exosome-mediated crosstalk and their resultant effects remain unexplored. OBJECTIVE: It is well established that exosome-mediated cellular communication can influence disease phenotypes. This study explores exosome-mediated cellular crosstalk between structural and immune cells in asthma pathogenesis. METHODS: Exosomes were isolated and detected from bronchoalveolar lavage fluid of control and asthmatic mice and were quantified by using a bead-based assay. Involvement of epithelial cells and macrophages were established by using immunohistochemical techniques in lung tissue sections. The role of IL-13 in exosome production was ascertained by using various in vitro and in vivo techniques. Exosome secretion was blocked in in vitro and in vivo settings by using a chemical inhibitor, and the effects on various asthmatic features were studied. RESULTS: Using combinatorial in vitro and in vivo approaches, we found that exosome secretion and production of exosome-associated proteins are higher in lungs of asthmatic mice compared with that seen in sham mice. Asthma is marked by enhanced secretion of exosomes by epithelial cells, but not macrophages, under the influence of IL-13. These epithelial cell exosomes induce proliferation and chemotaxis of undifferentiated macrophages. On the other hand, GW4869, which inhibited exosome production, resulted in a reduced population of proliferating monocytes and alleviation of various asthmatic features. CONCLUSION: Under the influence of IL-13, epithelial cell-derived exosomes can induce enhanced proliferation and chemotaxis of undifferentiated macrophages in the lungs during asthmatic inflammatory conditions. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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.)
Protein markers
EV: CD63/ CD81/ HSP70/ TSG101/ Annexin5
non-EV: None
Proteomics
no
EV density (g/ml)
1.16-1.21
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 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
Orientation
Top-down
Rotor type
SW50.1
Speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ HSP70/ TSG101/ Annexin5
ELISA
Detected EV-associated proteins
Annexin5
Characterization: Particle analysis
EV130043 1/2 Homo sapiens Cell culture supernatant dUC Kucharzewska P 2013 33%

Study summary

Full title
All authors
Kucharzewska P, Christianson HC, Welch JE, Svensson KJ, Fredlund E, Ringnér M, Mörgelin M, Bourseau-Guilmain E, Bengzon J, Belting M
Journal
Proc Natl Acad Sci U S A
Abstract
Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. Howeve (show more...)Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: CD63/ CD81/ Flotillin
non-EV: Cell organelle protein/ "Tubulin "
Proteomics
no
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ Flotillin
Detected contaminants
Cell organelle protein/ "Tubulin "
ELISA
Detected EV-associated proteins
Flotillin
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV130043 2/2 Homo sapiens Blood plasma dUC Kucharzewska P 2013 33%

Study summary

Full title
All authors
Kucharzewska P, Christianson HC, Welch JE, Svensson KJ, Fredlund E, Ringnér M, Mörgelin M, Bourseau-Guilmain E, Bengzon J, Belting M
Journal
Proc Natl Acad Sci U S A
Abstract
Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. Howeve (show more...)Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development. (hide)
EV-METRIC
33% (79th 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
dUC
Protein markers
EV: CD81/ "LAMP1 / CAV1"
non-EV: Cell organelle protein/ Tubulin
Proteomics
no
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 100,000 g and 150,000 g
Pelleting: time(min)
120
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ "LAMP1 / CAV1"
Detected contaminants
Cell organelle protein/ Tubulin
ELISA
Detected EV-associated proteins
"LAMP1 / CAV1"
Characterization: Particle analysis
NTA
EV130073 3/5 Homo sapiens Blood plasma 0.1 µm filter
dUC
Immunoaffinity
Kalra H 2013 33%

Study summary

Full title
All authors
Kalra H, Adda CG, Liem M, Ang CS, Mechler A, Simpson RJ, Hulett MD, Mathivanan S
Journal
Proteomics
Abstract
Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including b (show more...)Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active. (hide)
EV-METRIC
33% (79th 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
0.1 µm filter + dUC + Immunoaffinity
Protein markers
EV: Alix/ CD63/ CD9/ Flotilin1/ HSP70/ TSG101/ TFRC
non-EV: None
Proteomics
yes
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
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
Filtration steps
0.2µm > x > 0.1µm
Immunoaffinity capture
Selected surface protein(s)
EpCAM
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD9/ Flotilin1/ HSP70/ TSG101/ TFRC
ELISA
Detected EV-associated proteins
TFRC
Characterization: Particle analysis
EM
EM-type
transmission EM/ atomic force EM
Image type
Close-up, Wide-field
EV130073 4/5 Homo sapiens Blood plasma 0.1 µm filter
dUC
Kalra H 2013 33%

Study summary

Full title
All authors
Kalra H, Adda CG, Liem M, Ang CS, Mechler A, Simpson RJ, Hulett MD, Mathivanan S
Journal
Proteomics
Abstract
Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including b (show more...)Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active. (hide)
EV-METRIC
33% (79th 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
0.1 µm filter + dUC
Protein markers
EV: Alix/ CD63/ CD9/ Flotilin1/ HSP70/ TSG101/ TFRC
non-EV: None
Proteomics
yes
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
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
Wash: volume per pellet (ml)
10
Filtration steps
0.2µm > x > 0.1µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD9/ Flotilin1/ HSP70/ TSG101/ TFRC
ELISA
Detected EV-associated proteins
TFRC
Characterization: Particle analysis
EM
EM-type
transmission EM/ atomic force EM
Image type
Close-up, Wide-field
EV130011 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Kajimoto T 2013 33%

Study summary

Full title
All authors
Kajimoto T, Okada T, Miya S, Zhang L, Nakamura S
Journal
Nat Commun
Abstract
During late endosome maturation, cargo molecules are sorted into intralumenal vesicles (ILVs) of mul (show more...)During late endosome maturation, cargo molecules are sorted into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs), and are either delivered to lysosomes for degradation or fused with the plasma membranes for exosome release. The mechanism underlying formation of exosomal ILVs and cargo sorting into ILVs destined for exosome release is still unclear. Here we show that inhibitory G protein (Gi)-coupled sphingosine 1-phosphate (S1P) receptors regulate exosomal MVE maturation. Gi-coupled S1P receptors on MVEs are constitutively activated through a constant supply of S1P via autocrine activation within organelles. We also found that the continuous activation of Gi-coupled S1P receptors on MVEs is essential for cargo sorting into ILVs destined for exosome release. Our results reveal a mechanism underlying ESCRT-independent maturation of exosomal MVEs. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: CD63/ CD81/ HSP70/ Tf-receptor
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
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)
70
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ HSP70/ Tf-receptor
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Tf-receptor
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EV130144 2/2 Mus musculus Cell culture supernatant 0.2 µm filter
0.45 µm
dUC
Sucrose-DG (valid.)
Hassani K 2013 33%

Study summary

Full title
All authors
Hassani K, Olivier M
Journal
PLoS Negl Trop Dis
Abstract
Released by many eukaryotic cells, the exosomes are 40-100 nm vesicles shown to operate over the com (show more...)Released by many eukaryotic cells, the exosomes are 40-100 nm vesicles shown to operate over the complex processes of cell-cell communication. Among the metazoan cell lineages known to generate exosomes is the mononuclear phagocyte lineage, a lineage that parasites such as Leishmania are known to subvert as host cells. We previously reported that mouse macrophage signaling and functions are modified once co-incubated with exoproteome of Leishmania promastigotes. Using mass spectrometry analysis, we were curious to further compare the content of purified exosomes released by the J774 mouse macrophage cell line exposed or not to either LPS or to stationary phase Leishmania mexicana promastigotes. Collectively, our analyses resulted in detection of 248 proteins, ?50-80% of which were shared among the three sources studied. Using exponentially modified protein abundance index (emPAI) and network analyses, we found that the macrophage exosomes display unique signatures with respect to composition and abundance of many functional groups of proteins, such as plasma membrane-associated proteins, chaperones and metabolic enzymes. Moreover, for the first time, L. mexicana surface protease GP63 is shown to be present in exosomes released from J774 macrophages exposed to stationary phase promastigotes. We observed that macrophage exosomes are able to induce signaling molecules and transcription factors in naive macrophages. Finally, using qRT-PCR, we monitored modulation of expression of multiple immune-related genes within macrophages exposed to exosomes. We found all three groups of exosomes to induce expression of immune-related genes, the ones collected from macrophages exposed to L. mexicana sharing properties with exosomes collected from macrophage left unexposed to any agonist. Overall, our results allowed depicting that protein sorting into macrophage-derived exosomes depends upon the cell status and how such distinct protein sorting can in turn impact the functions of naive J774 cells. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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 + 0.45 µm + dUC + Sucrose-DG (valid.)
Protein markers
EV: Actin/ GP63/ Tubulin/ PGK1/ PABP
non-EV: None
Proteomics
yes
EV density (g/ml)
1.13-1.16
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 100,000 g and 150,000 g
Pelleting: time(min)
60
Density gradient
Only used for validation of main results
1
Highest density fraction
2
Orientation
Top-down
Speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Actin/ GP63/ Tubulin/ PGK1/ PABP
ELISA
Detected EV-associated proteins
Actin/ GP63/ Tubulin/ PGK1/ PABP
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV130090 1/2 Rattus norvegicus/rattus Cell culture supernatant dUC
Sucrose-DG (valid.)
Grapp M 2013 33%

Study summary

Full title
All authors
Grapp M, Wrede A, Schweizer M, Hüwel S, Galla HJ, Snaidero N, Simons M, Bückers J, Low PS, Urlaub H, Gärtner J, Steinfeld R
Journal
Nat Commun
Abstract
Loss of folate receptor-? function is associated with cerebral folate transport deficiency and child (show more...)Loss of folate receptor-? function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-cerebrospinal fluid barrier, we investigate the transport of 5-methyltetrahydrofolate in polarized cells. Here we identify folate receptor-?-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human folate receptor-?, and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both the apical medium of folate receptor-?-transfected rat choroid plexus cells and human cerebrospinal fluid contain folate receptor-?-positive exosomes. Loss of folate receptor-?-expressing cerebrospinal fluid exosomes correlates with severely reduced 5-methyltetrahydrofolate concentration, corroborating the importance of the folate receptor-?-mediated folate transport in the cerebrospinal fluid. Intraventricular injections of folate receptor-?-positive and -negative exosomes into mouse brains demonstrate folate receptor-?-dependent delivery of exosomes into the brain parenchyma. Our results unravel a new pathway of folate receptor-?-dependent exosome-mediated folate delivery into the brain parenchyma and opens new avenues for cerebral drug targeting. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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.)
Protein markers
EV: Alix/ Flotillin2
non-EV: None
Proteomics
yes
EV density (g/ml)
1.13-1.16
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
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)
150
Density gradient
Only used for validation of main results
1
Orientation
Top-down
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Flotillin2
ELISA
Detected EV-associated proteins
Flotillin2
Characterization: Particle analysis
EV130090 2/2 Homo sapiens "Cerebrospinal fluid" dUC
Sucrose-DG (valid.)
Grapp M 2013 33%

Study summary

Full title
All authors
Grapp M, Wrede A, Schweizer M, Hüwel S, Galla HJ, Snaidero N, Simons M, Bückers J, Low PS, Urlaub H, Gärtner J, Steinfeld R
Journal
Nat Commun
Abstract
Loss of folate receptor-? function is associated with cerebral folate transport deficiency and child (show more...)Loss of folate receptor-? function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-cerebrospinal fluid barrier, we investigate the transport of 5-methyltetrahydrofolate in polarized cells. Here we identify folate receptor-?-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human folate receptor-?, and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both the apical medium of folate receptor-?-transfected rat choroid plexus cells and human cerebrospinal fluid contain folate receptor-?-positive exosomes. Loss of folate receptor-?-expressing cerebrospinal fluid exosomes correlates with severely reduced 5-methyltetrahydrofolate concentration, corroborating the importance of the folate receptor-?-mediated folate transport in the cerebrospinal fluid. Intraventricular injections of folate receptor-?-positive and -negative exosomes into mouse brains demonstrate folate receptor-?-dependent delivery of exosomes into the brain parenchyma. Our results unravel a new pathway of folate receptor-?-dependent exosome-mediated folate delivery into the brain parenchyma and opens new avenues for cerebral drug targeting. (hide)
EV-METRIC
33% (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
"Cerebrospinal fluid"
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.)
Protein markers
EV: Flotillin2
non-EV: None
Proteomics
yes
EV density (g/ml)
1.11-1.16
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
"Cerebrospinal fluid"
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)
150
Density gradient
Only used for validation of main results
1
Orientation
Top-down
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin2
ELISA
Detected EV-associated proteins
Flotillin2
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV130026 1/3 Homo sapiens Urine dUC
UF
Gerlach JQ 2013 33%

Study summary

Full title
All authors
Gerlach JQ, Krüger A, Gallogly S, Hanley SA, Hogan MC, Ward CJ, Joshi L, Griffin MD
Journal
PLoS One
Abstract
Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomo (show more...)Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications. (hide)
EV-METRIC
33% (72nd 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
dUC + UF
Protein markers
EV: CD24/ AQP2
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 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)
150
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD24/ AQP2
ELISA
Detected EV-associated proteins
CD24/ AQP2
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Close-up
EV130075 3/3 Homo sapiens Urine dUC
Iodixanol-DG (valid.)
Fraser KB 2013 33%

Study summary

Full title
All authors
Fraser KB, Moehle MS, Daher JP, Webber PJ, Williams JY, Stewart CA, Yacoubian TA, Cowell RM, Dokland T, Ye T, Chen D, Siegal GP, Galemmo RA, Tsika E, Moore DJ, Standaert DG, Kojima K, Mobley JA, West AB
Journal
Hum Mol Genet
Abstract
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD) (show more...)Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials. (hide)
EV-METRIC
33% (72nd 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 + Iodixanol-DG (valid.)
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: None
Proteomics
yes
EV density (g/ml)
1.176
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Urine
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)
120
Density gradient
Only used for validation of main results
1
Speed (g)
500000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD9/ TSG101
Characterization: Particle analysis
EM
EM-type
cryo EM
Image type
Wide-field
EV130039 1/1 Homo sapiens Cell culture supernatant dUC
Iodixanol-DG
Feng Z 2013 33%

Study summary

Full title
All authors
Feng Z, Hensley L, McKnight KL, Hu F, Madden V, Ping L, Jeong SH, Walker C, Lanford RE, Lemon SM
Journal
Nature
Abstract
Animal viruses are broadly categorized structurally by the presence or absence of an envelope compos (show more...)Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver. Virus-specific antibodies appear only after 3-4 weeks of infection, and typically herald its resolution. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure, when virus replication is well established in the liver. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (eHAV) resemble exosomes, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT), namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between enveloped and non-enveloped viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
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 + Iodixanol-DG
Protein markers
EV: Alix/ Flotilin1
non-EV: None
Proteomics
no
EV density (g/ml)
1.100
Show all info
Study aim
Other/To decipher virus secretion into EVs
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
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
Lowest density fraction
8
Highest density fraction
40
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Flotilin1
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV130038 1/1 Homo sapiens Cell culture supernatant dUC Duijvesz D 2013 33%

Study summary

Full title
All authors
Duijvesz D, Burnum-Johnson KE, Gritsenko MA, Hoogland AM, Vredenbregt-van den Berg MS, Willemsen R, Luider T, Paša-Tolic L, Jenster G
Journal
PLoS One
Abstract
BACKGROUND: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biom (show more...)BACKGROUND: Current markers for prostate cancer, such as PSA lack specificity. Therefore, novel biomarkers are needed. Unfortunately, the complexity of body fluids often hampers biomarker discovery. An attractive alternative approach is the isolation of small vesicles, i.e. exosomes, ?100 nm, which contain proteins that are specific to the tissue from which they are derived and therefore can be considered as treasure chests for disease-specific biomarker discovery. MATERIALS AND METHODS: Exosomes were isolated from 2 immortalized primary prostate epithelial cells (PNT2C2 and RWPE-1) and 2 PCa cell lines (PC346C and VCaP) by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ-Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Candidate biomarkers were validated by Western blotting and Immunohistochemistry. RESULTS: Proteomic characterization resulted in the identification of 248, 233, 169, and 216 proteins by at least 2 peptides in exosomes from PNT2C2, RWPE-1, PC346C, and VCaP, respectively. Statistical analyses revealed 52 proteins differently abundant between PCa and control cells, 9 of which were more abundant in PCa. Validation by Western blotting confirmed a higher abundance of FASN, XPO1 and PDCD6IP (ALIX) in PCa exosomes. CONCLUSIONS: Identification of exosomal proteins using high performance LC-FTMS resulted in the discovery of PDCD6IP, FASN, XPO1 and ENO1 as new candidate biomarkers for prostate cancer. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: Alix/ CD9
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 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting: time(min)
110
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD9
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV130142 1/2 Homo sapiens Serum dUC
Immunoaffinity (valid.)
Sucrose-DG (valid.)
Di Noto G 2013 33%

Study summary

Full title
All authors
Di Noto G, Paolini L, Zendrini A, Radeghieri A, Caimi L, Ricotta D
Journal
PLoS One
Abstract
Plasma cell dyscrasias are immunosecretory disorders that can lead to hematological malignancies suc (show more...)Plasma cell dyscrasias are immunosecretory disorders that can lead to hematological malignancies such as Multiple Myeloma (MM). MM accounts for 15% of all hematologic cancers, and those diagnosed with MM typically become severely ill and have a low life expectancy. Monoclonal immunoglobulin Free Light Chains (FLC) are present in the serum and urine of many patients with plasma cell diseases. The biological differences between monoclonal FLCs, produced under malignant or benign dyscrasias, has not yet been characterized. In the present study, we show that endothelial and heart muscle cell lines internalize kappa and lambda FLCs. After internalization, FLCs are rerouted in the extracellular space via microvesicles and exosomes that can be re-internalized in contiguous cells. Only FLCs secreted from malignant B Lymphocytes were carried in Hsp70, annexin V, and c-src positive vesicles. In both MM and AL Amyloidosis patients we observed an increase in microvesicle and exosome production. Isolated serum vesicles from MM, AL Amyloidosis and monoclonal gammopathy of undetermined significance (MGUS) patients contained FLCs. Furthermore MM and AL amyloidosis vesicles were strongly positive for Hsp70, annexin V, and c-src compared to MGUS and control patients. These are the first data implying that FLCs reroute via microvesicles in the blood stream, and also suggest a potential novel mechanism of c-src activation in plasma cell dyscrasia. (hide)
EV-METRIC
33% (86th 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
microvesicles
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 + Immunoaffinity (valid.) + Sucrose-DG (valid.)
Adj. k-factor
89.21 (pelleting)
Protein markers
EV: HSP70/ Annexin5/ Tubulin
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
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
Pelleting: rotor type
TLA55
Pelleting: adjusted k-factor
89.21
Density gradient
Only used for validation of main results
1
Lowest density fraction
15
Highest density fraction
60
Orientation
Top-down
Immunoaffinity capture
Selected surface protein(s)
Annexin5
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
HSP70/ Annexin5/ Tubulin
ELISA
Detected EV-associated proteins
Annexin5/ Tubulin
Characterization: Particle analysis
EM
EM-type
scanning EM
Image type
Wide-field
EV130036 1/1 Homo sapiens Cell culture supernatant dUC Colombo M 2013 33%

Study summary

Full title
All authors
Colombo M, Moita C, van Niel G, Kowal J, Vigneron J, Benaroch P, Manel N, Moita LF, Théry C, Raposo G
Journal
J Cell Sci
Abstract
Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (M (show more...)Exosomes are extracellular vesicles (EVs) secreted upon fusion of endosomal multivesicular bodies (MVBs) with the plasma membrane. The mechanisms involved in their biogenesis have not yet been fully identified although they could be used to modulate exosome formation and therefore are a promising tool in understanding exosome functions. We have performed an RNA interference screen targeting 23 components of the endosomal sorting complex required for transport (ESCRT) machinery and associated proteins in MHC class II (MHC II)-expressing HeLa-CIITA cells. Silencing of HRS, STAM1 or TSG101 reduced the secretion of EV-associated CD63 and MHC II but each gene altered differently the size and/or protein composition of secreted EVs, as quantified by immuno-electron microscopy. By contrast, depletion of VPS4B augmented this secretion while not altering the features of EVs. For several other ESCRT subunits, it was not possible to draw any conclusions about their involvement in exosome biogenesis from the screen. Interestingly, silencing of ALIX increased MHC II exosomal secretion, as a result of an overall increase in intracellular MHC II protein and mRNA levels. In human dendritic cells (DCs), ALIX depletion also increased MHC II in the cells, but not in the released CD63-positive EVs. Such differences could be attributed to a greater heterogeneity in size, and higher MHC II and lower CD63 levels in vesicles recovered from DCs as compared with HeLa-CIITA. The results reveal a role for selected ESCRT components and accessory proteins in exosome secretion and composition by HeLa-CIITA. They also highlight biogenetic differences in vesicles secreted by a tumour cell line and primary DCs. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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 / 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
209.7 (pelleting)
Protein markers
EV: CD63/ CD81/ "Hsc70 / MHC2"
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
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 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
Pelleting: rotor type
45Ti
Pelleting: adjusted k-factor
209.7
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ "Hsc70 / MHC2"
ELISA
Detected EV-associated proteins
"Hsc70 / MHC2"
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV130086 2/5 Homo sapiens Pleural fluid 0.2 µm filter
dUC
Chugh PE 2013 33%

Study summary

Full title
All authors
Chugh PE, Sin SH, Ozgur S, Henry DH, Menezes P, Griffith J, Eron JJ, Damania B, Dittmer DP
Journal
PLoS Pathog
Abstract
MicroRNAs (miRNAs) are stable, small non-coding RNAs that modulate many downstream target genes. Rec (show more...)MicroRNAs (miRNAs) are stable, small non-coding RNAs that modulate many downstream target genes. Recently, circulating miRNAs have been detected in various body fluids and within exosomes, prompting their evaluation as candidate biomarkers of diseases, especially cancer. Kaposi's sarcoma (KS) is the most common AIDS-associated cancer and remains prevalent despite Highly Active Anti-Retroviral Therapy (HAART). KS is caused by KS-associated herpesvirus (KSHV), a gamma herpesvirus also associated with Primary Effusion Lymphoma (PEL). We sought to determine the host and viral circulating miRNAs in plasma, pleural fluid or serum from patients with the KSHV-associated malignancies KS and PEL and from two mouse models of KS. Both KSHV-encoded miRNAs and host miRNAs, including members of the miR-17-92 cluster, were detectable within patient exosomes and circulating miRNA profiles from KSHV mouse models. Further characterization revealed a subset of miRNAs that seemed to be preferentially incorporated into exosomes. Gene ontology analysis of signature exosomal miRNA targets revealed several signaling pathways that are known to be important in KSHV pathogenesis. Functional analysis of endothelial cells exposed to patient-derived exosomes demonstrated enhanced cell migration and IL-6 secretion. This suggests that exosomes derived from KSHV-associated malignancies are functional and contain a distinct subset of miRNAs. These could represent candidate biomarkers of disease and may contribute to the paracrine phenotypes that are a characteristic of KS. (hide)
EV-METRIC
33% (83rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
Pleural fluid
Focus 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
232.7 (pelleting) / 232.7 (washing)
Protein markers
EV: Flotillin2/ HSP90alpha/ HSP90beta/ Beta-actin
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Pleural fluid
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
SW32
Pelleting: adjusted k-factor
232.7
Wash: volume per pellet (ml)
35
Wash: Rotor Type
SW32
Wash: adjusted k-factor
232.7
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin2/ HSP90alpha/ HSP90beta/ Beta-actin
ELISA
Detected EV-associated proteins
Flotillin2/ HSP90alpha/ HSP90beta/ Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV130066 2/2 Bos bovis Epididymal fluid dUC Caballero JN 2013 33%

Study summary

Full title
All authors
Caballero JN, Frenette G, Belleannée C, Sullivan R
Journal
PLoS One
Abstract
Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the (show more...)Acquisition of fertilization ability by spermatozoa during epididymal transit occurs in part by the transfer of molecules from membranous vesicles called epididymosomes. Epididymosomes are heterogeneous in terms of both size and molecular composition. Exosomes and other related small membranous vesicles (30-120 nm) containing tetraspanin proteins on their surface are found in many biological fluids. In this study, we demonstrate that these vesicles are present in bovine cauda epididymal fluid as a subpopulation of epididymosomes. They contain tetraspanin CD9 in addition to other proteins involved in sperm maturation such as P25b, GliPr1L1, and MIF. In order to study the mechanism of protein transfer to sperm, DilC12-labeled unfractionated epididymosomes or CD9-positive microvesicles were coincubated with epididymal spermatozoa, and their transfer was evaluated by flow cytometry. CD9-positive microvesicles from epididymal fluid specifically transferred molecules to spermatozoa, whereas those prepared from blood were unable to do so. The CD9-positive microvesicles transferred molecules to the same sperm regions (acrosome and midpiece) as epididymosomes, with the same kinetics; however, the molecules were preferentially transferred to live sperm and, in contrast to epididymosomes, Zn(2+) did not demonstrate potentiated transfer. Tetraspanin CD9 was associated with other proteins on the membrane surface of CD9-positive microvesicles according to coimmunoprecipitation experiments. CD26 cooperated with CD9 in the molecular transfer to sperm since the amount of molecules transferred was significantly reduced in the presence of specific antibodies. In conclusion, CD9-positive microvesicles are present in bovine cauda epididymal fluid and transfer molecules to live maturing sperm in a tissue-specific manner that involves CD9 and CD26. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Epididymal fluid
Focus vesicles
microvesicles
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
Protein markers
EV: CD9
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Bos bovis
Sample Type
Epididymal fluid
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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Wide-field
EV130023 1/2 Drosophila melanogaster Cell culture supernatant dUC
Sucrose-DG (valid.)
Beckett K 2013 33%

Study summary

Full title
All authors
Beckett K, Monier S, Palmer L, Alexandre C, Green H, Bonneil E, Raposo G, Thibault P, Le Borgne R, Vincent JP
Journal
Traffic
Abstract
Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls gr (show more...)Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless-expressing Drosophila S2 cells, Wingless is present on exosome-like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless-containing exosome-like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome-like vesicles. Using these exosome markers and a cell-based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless-expressing cells. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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.)
Protein markers
EV: Flotillin2
non-EV: None
Proteomics
yes
EV density (g/ml)
1.17
Show all info
Study aim
Function
Sample
Species
Drosophila melanogaster
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
0.25
Highest density fraction
2
Orientation
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin2
ELISA
Detected EV-associated proteins
Flotillin2
Characterization: Particle analysis
EV130031 1/1 Mus musculus Cell culture supernatant dUC Basso M 2013 33%

Study summary

Full title
All authors
Basso M, Pozzi S, Tortarolo M, Fiordaliso F, Bisighini C, Pasetto L, Spaltro G, Lidonnici D, Gensano F, Battaglia E, Bendotti C, Bonetto V
Journal
J Biol Chem
Abstract
Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The me (show more...)Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
Protein markers
EV: Flotilin1/ VCP/p97/ SOD1
non-EV: None
Proteomics
no
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
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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ VCP/p97/ SOD1
ELISA
Detected EV-associated proteins
VCP/p97/ SOD1
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV130029 1/1 Homo sapiens Cell culture supernatant dUC Aqil M 2013 33%

Study summary

Full title
All authors
Aqil M, Naqvi AR, Bano AS, Jameel S
Journal
PLoS One
Abstract
The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membra (show more...)The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR). (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
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
253.9 (pelleting) / 253.9 (washing)
Protein markers
EV: Alix/ CD81/ TSG101/ GAPDH/ Beta-actin
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Other/HIV-1 accessory protein 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 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
SW28
Pelleting: adjusted k-factor
253.9
Wash: volume per pellet (ml)
30
Wash: Rotor Type
SW28
Wash: adjusted k-factor
253.9
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD81/ TSG101/ GAPDH/ Beta-actin
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
GAPDH/ Beta-actin
Characterization: Particle analysis
EV130134 1/1 Gram-negative bacteria Indoor dust 0.2 µm filter
0.45 µm
dUC
UF
Kim YS 2013 29%

Study summary

Full title
All authors
Kim YS, Choi EJ, Lee WH, Choi SJ, Roh TY, Park J, Jee YK, Zhu Z, Koh YY, Gho YS, Kim YK
Journal
Clin Exp Allergy
Abstract
BACKGROUND: Many bacterial components in indoor dust can evoke inflammatory pulmonary diseases. Bact (show more...)BACKGROUND: Many bacterial components in indoor dust can evoke inflammatory pulmonary diseases. Bacteria secrete nanometre-sized vesicles into the extracellular milieu, but it remains to be determined whether bacteria-derived extracellular vesicles in indoor dust are pathophysiologically related to inflammatory pulmonary diseases. OBJECTIVE: To evaluate whether extracellular vesicles (EV) in indoor air are related to the pathogenesis of pulmonary inflammation and/or asthma. METHODS: Indoor dust was collected from a bed mattress in an apartment. EV were prepared by sequential ultrafiltration and ultracentrifugation. Innate and adaptive immune responses were evaluated after airway exposure of EV. RESULTS: Repeated intranasal application of indoor-dust-induced neutrophilic pulmonary inflammation accompanied by lung infiltration of both Th1 and Th17 cells. EV 50-200 nm in diameter were present (102.5 ?g protein concentration/g dust) in indoor dust. These vesicles were internalized by airway epithelial cells and alveolar macrophages, and this process was blocked by treatment of polymyxin B (an antagonist of lipopolysaccharide, an outer-membrane component of Gram-negative bacteria). Intranasal application of 0.1 or 1 ?g of these vesicles for 4 weeks elicited neutrophilic pulmonary inflammation. This phenotype was accompanied by lung infiltration of both Th1 and Th17 cells, which were reversed by treatment of polymyxin B. Serum dust EV-reactive IgG1 levels were significantly higher in atopic children with asthma than in atopic healthy children and those with rhinitis or dermatitis. CONCLUSION & CLINICAL RELEVANCE: Indoor dust EV, especially derived from Gram-negative bacteria, is a possible causative agent of neutrophilic airway diseases. (hide)
EV-METRIC
29% (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
Indoor dust
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 + dUC + UF
Adj. k-factor
139.8 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Gram-negative bacteria
Sample Type
Indoor dust
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)
180
Pelleting: rotor type
45Ti
Pelleting: adjusted k-factor
139.8
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Wide-field
EV130076 2/3 Gut microbiota Mouse intestinal fluid 0.45 µm filter
dUC
Sucrose-DG (valid.)
Kang CS 2013 29%

Study summary

Full title
All authors
Kang CS, Ban M, Choi EJ, Moon HG, Jeon JS, Kim DK, Park SK, Jeon SG, Roh TY, Myung SJ, Gho YS, Kim JG, Kim YK
Journal
PLoS One
Abstract
Gut microbiota play an important part in the pathogenesis of mucosal inflammation, such as inflammat (show more...)Gut microbiota play an important part in the pathogenesis of mucosal inflammation, such as inflammatory bowel disease (IBD). However, owing to the complexity of the gut microbiota, our understanding of the roles of commensal and pathogenic bacteria in the maintenance of immune homeostasis in the gut is evolving only slowly. Here, we evaluated the role of gut microbiota and their secreting extracellular vesicles (EV) in the development of mucosal inflammation in the gut. Experimental IBD model was established by oral application of dextran sulfate sodium (DSS) to C57BL/6 mice. The composition of gut microbiota and bacteria-derived EV in stools was evaluated by metagenome sequencing using bacterial common primer of 16S rDNA. Metagenomics in the IBD mouse model showed that the change in stool EV composition was more drastic, compared to the change of bacterial composition. Oral DSS application decreased the composition of EV from Akkermansia muciniphila and Bacteroides acidifaciens in stools, whereas increased EV from TM7 phylum, especially from species DQ777900_s and AJ400239_s. In vitro pretreatment of A. muciniphila-derived EV ameliorated the production of a pro-inflammatory cytokine IL-6 from colon epithelial cells induced by Escherichia coli EV. Additionally, oral application of A. muciniphila EV also protected DSS-induced IBD phenotypes, such as body weight loss, colon length, and inflammatory cell infiltration of colon wall. Our data provides insight into the role of gut microbiota-derived EV in regulation of intestinal immunity and homeostasis, and A. muciniphila-derived EV have protective effects in the development of DSS-induced colitis. (hide)
EV-METRIC
29% (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
Mouse intestinal 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
0.45 µm filter + dUC + Sucrose-DG (valid.)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Gut microbiota
Sample Type
Mouse intestinal fluid
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
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)
120
Density gradient
Only used for validation of main results
1
Speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV130139 1/3 Homo sapiens "Cerebrospinal fluid" dUC Carone C 2013 29%

Study summary

Full title
All authors
Carone C, Genedani S, Leo G, Filaferro M, Fuxe K, Agnati LF
Journal
Mol Ther Nucleic Acids
Abstract
Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tum (show more...)Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.Molecular Therapy-Nucleic Acids (2013) 2, e109; doi:10.1038/mtna.2013.28; published online 23 July 2013. (hide)
EV-METRIC
29% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
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
"Cerebrospinal 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
dUC
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
"Cerebrospinal fluid"
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
80
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ cryo EM
Image type
Close-up, Wide-field
EV130132 1/1 Homo sapiens Cell culture supernatant 0.8 µm filter
dUC
Atai NA 2013 29%

Study summary

Full title
All authors
Atai NA, Balaj L, van Veen H, Breakefield XO, Jarzyna PA, Van Noorden CJ, Skog J, Maguire CA
Journal
J Neurooncol
Abstract
Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV (show more...)Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV binding and uptake into recipient cells may be of therapeutic value as well as enhance understanding of EV biology. Here, we show that heparin interacts with uptake of tumor-derived as well as non-tumor-derived EVs into recipient cells. Incubation of glioma cell-derived EVs with heparin resulted in micron-sized structures observed by transmission electron microscopy, with EVs clearly visible within these structures. Inclusion of heparin greatly diminished transfer of labeled EVs from donor to recipient tumor cells. We also show a direct interaction between heparin and EVs using confocal microscopy. We found that the block in EV uptake was at the level of cell binding and not internalization. Finally, incubation of glioma-derived EVs containing EGFRvIII mRNA with he