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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
EV110001 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG
UF
Atay S 2011 78%

Study summary

Full title
All authors
Atay S, Gercel-Taylor C, Kesimer M, Taylor DD
Journal
Exp Cell Res
Abstract
Exosomes represent an important intercellular communication vehicle, mediating events essential for (show more...)Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165nm ± 0.5nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of normal early pregnancy. (hide)
EV-METRIC
78% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
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 + UF
Protein markers
EV: Alix/ CD81/ HSP70/ Beta-actin/ ICAM1/ HSP60
non-EV: Cell organelle protein
Proteomics
yes
EV density (g/ml)
1.13-1.17
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)
90
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Rotor type
90Ti
Speed (g)
105000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD81/ HSP70/ Beta-actin/ ICAM1/ HSP60
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Beta-actin/ ICAM1/ HSP60
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV110003 1/1 Homo sapiens
Mus musculus
Cell culture supernatant dUC
Sucrose-DG
Valapala M 2011 67%

Study summary

Full title
All authors
Valapala M, Vishwanatha JK
Journal
J Biol Chem
Abstract
Annexin A2 (AnxA2), a Ca(2+)-dependent phospholipid-binding protein, is known to associate with the (show more...)Annexin A2 (AnxA2), a Ca(2+)-dependent phospholipid-binding protein, is known to associate with the plasma membrane and the endosomal system. Within the plasma membrane, AnxA2 associates in a Ca(2+) dependent manner with cholesterol-rich lipid raft microdomains. Here, we show that the association of AnxA2 with the lipid rafts is influenced not only by intracellular levels of Ca(2+) but also by N-terminal phosphorylation at tyrosine 23. Binding of AnxA2 to the lipid rafts is followed by the transport along the endocytic pathway to be associated with the intralumenal vesicles of the multivesicular endosomes. AnxA2-containing multivesicular endosomes fuse directly with the plasma membrane resulting in the release of the intralumenal vesicles into the extracellular environment, which facilitates the exogenous transfer of AnxA2 from one cell to another. Treatment with Ca(2+) ionophore triggers the association of AnxA2 with the specialized microdomains in the exosomal membrane that possess raft-like characteristics. Phosphorylation at Tyr-23 is also important for the localization of AnxA2 to the exosomal membranes. These results suggest that AnxA2 is trafficked from the plasma membrane rafts and is selectively incorporated into the lumenal membranes of the endosomes to escape the endosomal degradation pathway. The Ca(2+)-dependent exosomal transport constitutes a novel pathway of extracellular transport of AnxA2. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Adj. k-factor
226 (pelleting) / 226 (washing)
Protein markers
EV: CD63/ CD81/ HSP70/ LAMP1/ Tf-receptor
non-EV: Cell organelle protein/ Annexin2
Proteomics
no
EV density (g/ml)
1.08-1.16
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Homo sapiens / Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
900
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
226
Wash: Rotor Type
SW28
Wash: adjusted k-factor
226
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ HSP70/ LAMP1/ Tf-receptor
Detected contaminants
Cell organelle protein/ Annexin2
ELISA
Detected EV-associated proteins
LAMP1/ Tf-receptor
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV110002 1/1 Homo sapiens
Canis familiaris
Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Higginbotham JN 2011 67%

Study summary

Full title
All authors
Higginbotham JN, Demory Beckler M, Gephart JD, Franklin JL, Bogatcheva G, Kremers GJ, Piston DW, Ayers GD, McConnell RE, Tyska MJ, Coffey RJ
Journal
Curr Biol
Abstract
Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands inclu (show more...)Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-? (TGF?), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGF? or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGF? or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG (valid.)
Protein markers
EV: CD9/ Flotilin1/ HSP70/ TSG101/ ADAM10/ ADAM17
non-EV: Cell organelle protein
Proteomics
no
TEM measurements
51.3(median)
Show all info
Study aim
Function
Sample
Species
Homo sapiens / Canis familiaris
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)
150
Density gradient
Only used for validation of main results
1
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ Flotilin1/ HSP70/ TSG101/ ADAM10/ ADAM17
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
ADAM10/ ADAM17
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV110020 2/3 Homo sapiens Serum Density cushion
Immunoaffinity (valid.)
Sucrose-DG (valid.)
Rupp AK 2011 63%

Study summary

Full title
All authors
Rupp AK, Rupp C, Keller S, Brase JC, Ehehalt R, Fogel M, Moldenhauer G, Marmé F, Sültmann H, Altevogt P
Journal
Gynecol Oncol
Abstract
OBJECTIVE: Cancer cells in the body release soluble and membranous factors that manipulate the tumor (show more...)OBJECTIVE: Cancer cells in the body release soluble and membranous factors that manipulate the tumor environment to facilitate growth and survival. Recent years have provided evidence that small microvesicles that are termed exosomes may play a pivotal role in this process. Exosomes are membrane vesicles with a size of 40-100 nm that are released by both tumor and normal cells and can be found in various body fluids. Tumor-derived exosomes carry functional proteins, mRNAs, and miRNAs and could serve as novel platform for tumor diagnosis and prognosis. However, marker proteins that allow enrichment of tumor-derived exosomes over normal exosomes are less well defined. METHODS: We used Western blot analysis and antibody coupled magnetic beads to characterize CD24 and EpCAM as markers for exosomes. We investigated ovarian carcinoma ascites, pleural effusions and serum of breast carcinoma patients. As non-tumor derived control we used exosomes from ascites of liver cirrhosis patients. RESULTS: Exosomes could be isolated from all body fluids and contained marker proteins as well as miRNAs. We observed that CD24 and EpCAM were selectively present on ascites exosomes of tumor patients and copurified together on anti-EpCAM or anti-CD24 magnetic beads. In breast cancer patients CD24 was present but EpCAM was absent from serum exosomes. Instead, the intact EpCAM ectodomain was recovered in a soluble form. We provide evidence that EpCAM can be cleaved from exosomes via serum metalloproteinase(s). CONCLUSION: Loss of EpCAM on serum exosomes may hamper enrichment by immune-affinity isolation. We suggest that CD24 could be an additional marker for the enrichment of tumor-derived exosomes from blood. (hide)
EV-METRIC
63% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density cushion + Immunoaffinity (valid.) + Sucrose-DG (valid.)
Protein markers
EV: CD9/ ADAM10/ CD24
non-EV: EpCAM
Proteomics
no
EV density (g/ml)
1.04-1.14
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Serum
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
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Immunoaffinity capture
Selected surface protein(s)
EpCAM, CD24
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ ADAM10/ CD24
Detected contaminants
EpCAM
ELISA
Detected EV-associated proteins
ADAM10/ CD24
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV110006 1/1 Drosophila melanogaster Drosophila Density cushion
Sucrose-DG (valid.)
Koppen T 2011 63%

Study summary

Full title
All authors
Koppen T, Weckmann A, Müller S, Staubach S, Bloch W, Dohmen RJ, Schwientek T
Journal
Proteomics
Abstract
Distinct types of vesicles are formed in eukaryotic cells that conduct a variable set of functions d (show more...)Distinct types of vesicles are formed in eukaryotic cells that conduct a variable set of functions depending on their origin. One subtype designated circulating microvesicles (MVs) provides a novel form of intercellular communication and recent work suggested the release and uptake of morphogens in vesicles by Drosophila cells. In this study, we have examined cells of the hemocyte-like cell lines Kc167 and S2 and identified secreted vesicles in the culture supernatant. The vesicles were isolated and found to have characteristics comparable to exosomes and plasma membrane MVs released by mammalian cells. In wingless-transfected cells, the full-length protein was detected in the vesicle isolates. Proteomics analyses of the vesicles identified 269 proteins that include various orthologs of marker proteins and proteins with putative functions in vesicle formation and release. Analogous to their mammalian counterparts, the subcellular origin of the vesicular constituents of both cell lines is dominated by membrane-associated and cytosolic proteins with functions that are consistent with their localization in MVs. The analyses revealed a significant overlap of the Kc167 and S2 vesicle proteomes and confirmed a close correlation with non-mammalian and mammalian exosomes. (hide)
EV-METRIC
63% (87th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Drosophila
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
Density cushion + Sucrose-DG (valid.)
Protein markers
EV: Rho1/ Rop/ Beta-tubulin
non-EV: None
Proteomics
yes
EV density (g/ml)
1.16-1.21
Show all info
Study aim
Omics
Sample
Species
Drosophila melanogaster
Sample Type
Drosophila
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
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Rotor type
TLA55
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Rho1/ Rop/ Beta-tubulin
ELISA
Detected EV-associated proteins
Rho1/ Rop/ Beta-tubulin
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV110004 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG
Wang GH 2011 56%

Study summary

Full title
All authors
Wang GH, Zhou XM, Bai Y, Yin XM, Yang LF, Zhao D
Journal
Cell Biol Int
Abstract
PrPC (cellular prion protein) is a GPI (glycophosphatidylinositol)-anchored protein present on the s (show more...)PrPC (cellular prion protein) is a GPI (glycophosphatidylinositol)-anchored protein present on the surface of a number of peripheral blood cells. PrPC must be present for the generation and propagation of pathogenic conformer [PrPSc (scrapie prion protein)], which is a conformational conversion form of PrPC and has a central role in transmissible spongiform encephalopathies. It is important to determine the transportation mechanism of normal PrPC between cells. Exosomes are membrane vesicles released into the extracellular space upon fusion of multivesicular endosomes with the plasma membrane. We have identified that THP-1 monocytes can secrete exosomes to culture medium, and the secreted exosomes can bear PrPC. We also found that Hsp70 interacts with PrPC not only in intracellular environment, but in the secreted exosomes. However, the specific markers of exosomes, Tsg101 and flotillin-1, were found with no interaction with PrPC. Our results demonstrated that PrPC can be released from THP-1 monocytes via secreted exosomes, and in this process, Hsp70 binds to PrPC, which suggests that Hsp70 may play a potential functional role in the release of PrPC. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Protein markers
EV: Flotilin1/ HSP70/ TSG101
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.13-1.16
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
Density gradient
Lowest density fraction
0.25
Highest density fraction
2.25
Orientation
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Wide-field
EV110011 3/3 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
Stamer WD 2011 56%

Study summary

Full title
All authors
Stamer WD, Hoffman EA, Luther JM, Hachey DL, Schey KL
Journal
J Proteomics
Abstract
To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular p (show more...)To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Adj. k-factor
255.8 (pelleting)
Protein markers
EV: CD81/ Flotilin1/ Syntenin/ Annexin2/ Annexin5/ GAPDH
non-EV: None
Proteomics
yes
EV density (g/ml)
1.100
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
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW41
Pelleting: adjusted k-factor
255.8
Wash: volume per pellet (ml)
10
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
CD81/ Flotilin1/ Syntenin/ Annexin2/ Annexin5/ GAPDH
ELISA
Detected EV-associated proteins
Annexin2/ Annexin5/ GAPDH
Characterization: Particle analysis
EV110010 1/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Hasegawa T 2011 56%

Study summary

Full title
All authors
Hasegawa T, Konno M, Baba T, Sugeno N, Kikuchi A, Kobayashi M, Miura E, Tanaka N, Tamai K, Furukawa K, Arai H, Mori F, Wakabayashi K, Aoki M, Itoyama Y, Takeda A
Journal
PLoS One
Abstract
Many neurodegenerative diseases share a common pathological feature: the deposition of amyloid-like (show more...)Many neurodegenerative diseases share a common pathological feature: the deposition of amyloid-like fibrils composed of misfolded proteins. Emerging evidence suggests that these proteins may spread from cell-to-cell and encourage the propagation of neurodegeneration in a prion-like manner. Here, we demonstrated that ?-synuclein (?SYN), a principal culprit for Lewy pathology in Parkinson's disease (PD), was present in endosomal compartments and detectably secreted into the extracellular milieu. Unlike prion protein, extracellular ?SYN was mainly recovered in the supernatant fraction rather than in exosome-containing pellets from the neuronal culture medium and cerebrospinal fluid. Surprisingly, impaired biogenesis of multivesicular body (MVB), an organelle from which exosomes are derived, by dominant-negative mutant vacuolar protein sorting 4 (VPS4) not only interfered with lysosomal targeting of ?SYN but facilitated ?SYN secretion. The hypersecretion of ?SYN in VPS4-defective cells was efficiently restored by the functional disruption of recycling endosome regulator Rab11a. Furthermore, both brainstem and cortical Lewy bodies in PD were found to be immunoreactive for VPS4. Thus, VPS4, a master regulator of MVB sorting, may serve as a determinant of lysosomal targeting or extracellular secretion of ?SYN and thereby contribute to the intercellular propagation of Lewy pathology in PD. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG (valid.)
Adj. k-factor
279.8 (pelleting)
Protein markers
EV: Alix/ Flotilin1/ HSP90/ PrP
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
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
Pelleting: rotor type
P40ST
Pelleting: adjusted k-factor
279.8
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Flotilin1/ HSP90/ PrP
Detected contaminants
Albumin
ELISA
Detected EV-associated proteins
PrP
Characterization: Particle analysis
EV110020 3/3 Homo sapiens Ascites Density cushion
Immunoaffinity (valid.)
Sucrose-DG (valid.)
Rupp AK 2011 50%

Study summary

Full title
All authors
Rupp AK, Rupp C, Keller S, Brase JC, Ehehalt R, Fogel M, Moldenhauer G, Marmé F, Sültmann H, Altevogt P
Journal
Gynecol Oncol
Abstract
OBJECTIVE: Cancer cells in the body release soluble and membranous factors that manipulate the tumor (show more...)OBJECTIVE: Cancer cells in the body release soluble and membranous factors that manipulate the tumor environment to facilitate growth and survival. Recent years have provided evidence that small microvesicles that are termed exosomes may play a pivotal role in this process. Exosomes are membrane vesicles with a size of 40-100 nm that are released by both tumor and normal cells and can be found in various body fluids. Tumor-derived exosomes carry functional proteins, mRNAs, and miRNAs and could serve as novel platform for tumor diagnosis and prognosis. However, marker proteins that allow enrichment of tumor-derived exosomes over normal exosomes are less well defined. METHODS: We used Western blot analysis and antibody coupled magnetic beads to characterize CD24 and EpCAM as markers for exosomes. We investigated ovarian carcinoma ascites, pleural effusions and serum of breast carcinoma patients. As non-tumor derived control we used exosomes from ascites of liver cirrhosis patients. RESULTS: Exosomes could be isolated from all body fluids and contained marker proteins as well as miRNAs. We observed that CD24 and EpCAM were selectively present on ascites exosomes of tumor patients and copurified together on anti-EpCAM or anti-CD24 magnetic beads. In breast cancer patients CD24 was present but EpCAM was absent from serum exosomes. Instead, the intact EpCAM ectodomain was recovered in a soluble form. We provide evidence that EpCAM can be cleaved from exosomes via serum metalloproteinase(s). CONCLUSION: Loss of EpCAM on serum exosomes may hamper enrichment by immune-affinity isolation. We suggest that CD24 could be an additional marker for the enrichment of tumor-derived exosomes from blood. (hide)
EV-METRIC
50% (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
Ascites
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
Density cushion + Immunoaffinity (valid.) + Sucrose-DG (valid.)
Protein markers
EV: CD9/ HSP70/ EpCAM/ ADAM10/ Annexin1/ CD24
non-EV: None
Proteomics
no
EV density (g/ml)
1.04-1.14
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Ascites
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
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Immunoaffinity capture
Selected surface protein(s)
EpCAM, CD24
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ HSP70/ EpCAM/ ADAM10/ Annexin1/ CD24
ELISA
Detected EV-associated proteins
EpCAM/ ADAM10/ Annexin1/ CD24
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV110019 1/1 Homo sapiens Urine dUC
Sucrose-DG
Moon PG 2011 44%

Study summary

Full title
All authors
Moon PG, Lee JE, You S, Kim TK, Cho JH, Kim IS, Kwon TH, Kim CD, Park SH, Hwang D, Kim YL, Baek MC
Journal
Proteomics
Abstract
To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement memb (show more...)To identify biomarker candidates associated with early IgA nephropathy (IgAN) and thin basement membrane nephropathy (TBMN), the most common causes presenting isolated hematuria in childhood, a proteomic approach of urinary exosomes from early IgAN and TBMN patients was introduced. The proteomic results from the patients were compared with a normal group to understand the pathophysiological processes associated with these diseases at the protein level. The urinary exosomes, which reflect pathophysiological processes, collected from three groups of young adults (early IgAN, TBMN, and normal) were trypsin-digested using a gel-assisted protocol, and quantified by label-free LC-MS/MS, using an MS(E) mode. A total of 1877 urinary exosome proteins, including cytoplasmic, membrane, and vesicle trafficking proteins, were identified. Among the differentially expressed proteins, four proteins (aminopeptidase N, vasorin precursor, ?-1-antitrypsin, and ceruloplasmin) were selected as biomarker candidates to differentiate early IgAN from TBMN. We confirmed the protein levels of the four biomarker candidates by semi-quantitative immunoblot analysis in urinary exosomes independently prepared from other patients, including older adult groups. Further clinical studies are needed to investigate the diagnostic and prognostic value of these urinary markers for early IgAN and TBMN. Taken together, this study showed the possibility of identifying biomarker candidates for human urinary diseases using urinary exosomes and might help to understand the pathophysiology of early IgAN and TBMN at the protein level. (hide)
EV-METRIC
44% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
Adj. k-factor
69.34 (pelleting)
Protein markers
EV: CD63/ CD9/ NHE3
non-EV: Cell organelle protein
Proteomics
yes
Show all info
Study aim
Biomarker
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
Pelleting: rotor type
SW55
Pelleting: adjusted k-factor
69.34
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Speed (g)
200000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9/ NHE3
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
NHE3
Characterization: Particle analysis
EV110027 2/2 Rattus norvegicus/rattus Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Lachenal G 2011 44%

Study summary

Full title
All authors
Lachenal G, Pernet-Gallay K, Chivet M, Hemming FJ, Belly A, Bodon G, Blot B, Haase G, Goldberg Y, Sadoul R
Journal
Mol Cell Neurosci
Abstract
Exosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane (show more...)Exosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane of endosomal intermediates called multivesicular bodies. They represent ways for discarding proteins and metabolites and also for intercellular transfer of proteins and RNAs. In the nervous system, it has been hypothesized that exosomes might be involved in the normal physiology of the synapse and possibly allow the trans-synaptic propagation of pathogenic proteins throughout the tissue. As a first step to validate this concept, we used biochemical and morphological approaches to demonstrate that mature cortical neurons in culture do indeed secrete exosomes. Using electron microscopy, we observed exosomes being released from somato-dendritic compartments. The endosomal origin of exosomes was demonstrated by showing that the C-terminal domain of tetanus toxin specifically endocytosed by neurons and accumulating inside multivesicular bodies, is released in the extracellular medium in association with exosomes. Finally, we found that exosomal release is modulated by glutamatergic synaptic activity, suggesting that this process might be part of normal synaptic physiology. Thus, our study paves the way towards the demonstration that exosomes take part in the physiology of the normal and pathological nervous system. (hide)
EV-METRIC
44% (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
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: Alix/ Flotilin1/ L1CAM/ GluR2/3
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.15
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus/rattus
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)
50
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.21099999999999999
Highest density fraction
2.2549999999999999
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Flotilin1/ L1CAM/ GluR2/3
ELISA
Detected EV-associated proteins
L1CAM/ GluR2/3
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV110018 1/3 Homo sapiens Saliva dUC
Sucrose-DG (valid.)
Keller S 2011 44%

Study summary

Full title
All authors
Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P
Journal
J Transl Med
Abstract
BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by diffe (show more...)BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis. METHOD: We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism. RESULTS: Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product. CONCLUSIONS: Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics. (hide)
EV-METRIC
44% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Saliva
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: CD9/ CD24/ Annexin1/ ADAM10
non-EV: None
Proteomics
no
EV density (g/ml)
1.08-1.14
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Saliva
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
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
CD9/ CD24/ Annexin1/ ADAM10
ELISA
Detected EV-associated proteins
CD24/ Annexin1/ ADAM10
Characterization: Particle analysis
EV110018 2/3 Homo sapiens Amniotic fluid dUC
Sucrose-DG (valid.)
Keller S 2011 44%

Study summary

Full title
All authors
Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P
Journal
J Transl Med
Abstract
BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by diffe (show more...)BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis. METHOD: We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism. RESULTS: Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product. CONCLUSIONS: Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics. (hide)
EV-METRIC
44% (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
Amniotic 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: CD9/ CD24/ Annexin1/ ADAM10
non-EV: None
Proteomics
no
EV density (g/ml)
1.08-1.14
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Amniotic fluid
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
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
CD9/ CD24/ Annexin1/ ADAM10
ELISA
Detected EV-associated proteins
CD24/ Annexin1/ ADAM10
Characterization: Particle analysis
EV110018 3/3 Homo sapiens Urine dUC
Sucrose-DG (valid.)
Keller S 2011 44%

Study summary

Full title
All authors
Keller S, Ridinger J, Rupp AK, Janssen JW, Altevogt P
Journal
J Transl Med
Abstract
BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by diffe (show more...)BACKGROUND: Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis. METHOD: We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism. RESULTS: Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product. CONCLUSIONS: Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics. (hide)
EV-METRIC
44% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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: CD9/ HSP70/ CD24/ ADAM10
non-EV: None
Proteomics
no
EV density (g/ml)
1.08-1.14
Show all info
Study aim
Biomarker
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
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
CD9/ HSP70/ CD24/ ADAM10
ELISA
Detected EV-associated proteins
CD24/ ADAM10
Characterization: Particle analysis
EV110038 3/3 Rattus norvegicus/rattus Cell culture supernatant dUC
Sucrose-DG (valid.)
Fitzner D 2011 44%

Study summary

Full title
All authors
Fitzner D, Schnaars M, van Rossum D, Krishnamoorthy G, Dibaj P, Bakhti M, Regen T, Hanisch UK, Simons M
Journal
J Cell Sci
Abstract
The transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenes (show more...)The transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenesis of autoimmune diseases. Here, we show that oligodendrocytes secrete small membrane vesicles called exosomes, which are specifically and efficiently taken up by microglia both in vitro and in vivo. Internalisation of exosomes occurs by a macropinocytotic mechanism without inducing a concomitant inflammatory response. After stimulation of microglia with interferon-?, we observe an upregulation of MHC class II in a subpopulation of microglia. However, exosomes are preferentially internalised in microglia that do not seem to have antigen-presenting capacity. We propose that the constitutive macropinocytotic clearance of exosomes by a subset of microglia represents an important mechanism through which microglia participate in the degradation of oligodendroglial membrane in an immunologically 'silent' manner. By designating the capacity for macropinocytosis and antigen presentation to distinct cells, degradation and immune function might be assigned to different subtypes of microglia. (hide)
EV-METRIC
44% (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
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/ TSG101/ Flotillin2/ PLP/ MOG
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.150
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Rattus norvegicus/rattus
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)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
1.03g/cm3
Highest density fraction
1.32g/cm3
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ Flotillin2/ PLP/ MOG
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Flotillin2/ PLP/ MOG
Characterization: Particle analysis
EV110005 1/1 Rattus norvegicus/rattus Cell culture supernatant dUC
Sucrose-DG (valid.)
Carayon K 2011 44%

Study summary

Full title
All authors
Carayon K, Chaoui K, Ronzier E, Lazar I, Bertrand-Michel J, Roques V, Balor S, Terce F, Lopez A, Salomé L, Joly E
Journal
J Biol Chem
Abstract
During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large (show more...)During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process. (hide)
EV-METRIC
44% (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
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: HSP90/ TSG101/ HSC70/ CHMP4B/ CHMP4C/ MHC1
non-EV: None
Proteomics
yes
EV density (g/ml)
1.16-1.21
Show all info
Study aim
Omics
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
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
0.5
Highest density fraction
2.5
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
HSP90/ TSG101/ HSC70/ CHMP4B/ CHMP4C/ MHC1
ELISA
Detected EV-associated proteins
HSC70/ CHMP4B/ CHMP4C/ MHC1
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV110026 2/4 Homo sapiens Saliva dUC Berckmans RJ 2011 44%

Study summary

Full title
All authors
Berckmans RJ, Sturk A, van Tienen LM, Schaap MC, Nieuwland R
Journal
Blood
Abstract
On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, (show more...)On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds. (hide)
EV-METRIC
44% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Saliva
Focus vesicles
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
472.2 (pelleting)
Protein markers
EV: TF
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Saliva
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
30
Pelleting: rotor type
TLA55
Pelleting: adjusted k-factor
472.2
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TF
ELISA
Detected EV-associated proteins
TF
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV110026 4/4 Homo sapiens Saliva dUC Berckmans RJ 2011 44%

Study summary

Full title
All authors
Berckmans RJ, Sturk A, van Tienen LM, Schaap MC, Nieuwland R
Journal
Blood
Abstract
On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, (show more...)On vascular damage, coagulation is initiated by extravascular tissue factor (TF). Intravascular TF, which is present on circulating cell-derived vesicles, is noncoagulant under physiologic conditions but prothrombotic under pathologic conditions. Human saliva triggers coagulation, but the mechanism and physiologic relevance are unknown. Because saliva is known to contain TF, we hypothesized that this TF may also be associated with cell-derived vesicles to facilitate coagulation when saliva directly contacts blood. The saliva-induced shortening of the clotting time of autologous plasma and whole blood from healthy subjects (n = 10) proved TF-dependent. This TF was associated with various types of cell-derived vesicles, including microparticles and exosomes. The physiologic function was shown by adding saliva to human pericardial wound blood collected from patients undergoing cardiac surgery. Addition of saliva shortened the clotting time from 300 ± 96 to 186 ± 24 seconds (P = .03). Our results show that saliva triggers coagulation, thereby reducing blood loss and the risk of pathogens entering the blood. We postulate that our reflex to lick a wound may be a mechanism to enable TF-exposing vesicles, present in saliva, to aid in the coagulation process and thus protect the organism from entering pathogens. This unique compartmentalization may be highly conserved because also animals lick their wounds. (hide)
EV-METRIC
44% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Saliva
Focus vesicles
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
57.93 (pelleting)
Protein markers
EV: TF
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Saliva
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
TLA55
Pelleting: adjusted k-factor
57.93
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TF
ELISA
Detected EV-associated proteins
TF
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV110025 1/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Aung T 2011 44%

Study summary

Full title
All authors
Aung T, Chapuy B, Vogel D, Wenzel D, Oppermann M, Lahmann M, Weinhage T, Menck K, Hupfeld T, Koch R, Trümper L, Wulf GG
Journal
Proc Natl Acad Sci U S A
Abstract
Targeting the surface of malignant cells has evolved into a cornerstone in cancer therapy, paradigma (show more...)Targeting the surface of malignant cells has evolved into a cornerstone in cancer therapy, paradigmatically introduced by the success of humoral immunotherapy against CD20 in malignant lymphoma. However, tumor cell susceptibility to immunochemotherapy varies, with mostly a fatal outcome in cases of resistant disease. Here, we show that lymphoma exosomes shield target cells from antibody attack and that exosome biogenesis is modulated by the lysosome-related organelle-associated ATP-binding cassette (ABC) transporter A3 (ABCA3). B-cell lymphoma cells released exosomes that carried CD20, bound therapeutic anti-CD20 antibodies, consumed complement, and protected target cells from antibody attack. ABCA3, previously shown to mediate resistance to chemotherapy, was critical for the amounts of exosomes released, and both pharmacological blockade and the silencing of ABCA3 enhanced susceptibility of target cells to antibody-mediated lysis. Mechanisms of cancer cell resistance to drugs and antibodies are linked in an ABCA3-dependent pathway of exosome secretion. (hide)
EV-METRIC
44% (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
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
213.3 (pelleting)
Protein markers
EV: Alix/ CD63/ CD9/ TSG101/ Flotillin2/ CD20
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
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)
240
Pelleting: rotor type
32Ti
Pelleting: adjusted k-factor
213.3
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD9/ TSG101/ Flotillin2/ CD20
ELISA
Detected EV-associated proteins
Flotillin2/ CD20
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV110067 2/3 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Vallhov H 2011 43%

Study summary

Full title
All authors
Vallhov H, Gutzeit C, Johansson SM, Nagy N, Paul M, Li Q, Friend S, George TC, Klein E, Scheynius A, Gabrielsson S
Journal
J Immunol
Abstract
Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes (show more...)Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV(-) B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350-CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression. (hide)
EV-METRIC
43% (82nd 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: MHC2/ CD23
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
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
Between 50,000 g and 100,000 g
Pelleting: time(min)
60
Filtration steps
0.22µm or 0.2µm
Western Blot
Detected EV-associated proteins
MHC2/ CD23
ELISA
Detected EV-associated proteins
MHC2/ CD23
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
NTA
Particle analysis: flow cytometry
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV110058 1/1 Homo sapiens Cell culture supernatant dUC
Iodixanol-DG
Ruiss R 2011 43%

Study summary

Full title
All authors
Ruiss R, Jochum S, Mocikat R, Hammerschmidt W, Zeidler R
Journal
PLoS One
Abstract
gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by inte (show more...)gp350, the major envelope protein of Epstein-Barr-Virus, confers B-cell tropism to the virus by interacting with the B lineage marker CD21. Here we utilize gp350 to generate tailored exosomes with an identical tropism. These exosomes can be used for the targeted co-transfer of functional proteins to normal and malignant human B cells. We demonstrate here the co-transfer of functional CD154 protein on tailored gp350+ exosomes to malignant B blasts from patients with B chronic lymphocytic leukemia (B-CLL), rendering B blasts immunogenic to tumor-reactive autologous T cells. Intriguingly, engulfment of gp350+ exosomes by B-CLL cells and presentation of gp350-derived peptides also re-stimulated EBV-specific T cells and redirected the strong antiviral cellular immune response in patients to leukemic B cells. In essence, we show that gp350 alone confers B-cell tropism to exosomes and that these exosomes can be further engineered to simultaneously trigger virus- and tumor-specific immune responses. The simultaneous exploitation of gp350 as a tropism molecule for tailored exosomes and as a neo-antigen in malignant B cells provides a novel attractive strategy for immunotherapy of B-CLL and other B-cell malignancies. (hide)
EV-METRIC
43% (82nd 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 + Iodixanol-DG
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.03-1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
253.9
Density gradient
Lowest density fraction
5
Highest density fraction
30
Characterization: Particle analysis
EV110050 1/2 Rattus norvegicus/rattus Cell culture supernatant dUC
Immunoaffinity (valid.)
Sucrose-DG (valid.)
Müller G 2011 43%

Study summary

Full title
All authors
Müller G, Schneider M, Biemer-Daub G, Wied S
Journal
Cell Signal
Abstract
Small microvesicles, such as microparticles and exosomes, have been demonstrated to transfer protein (show more...)Small microvesicles, such as microparticles and exosomes, have been demonstrated to transfer proteins and nucleic acids from a variety of donor to acceptor cells with corresponding (patho)physiological consequences. Recently the in vitro transfer of glycosylphosphatidylinositol (GPI)-anchored proteins from microvesicles released from large rat adipocytes to intracellular lipid droplets (LDs) of small adipocytes has been shown to be upregulated by physiological (palmitate, H(2)O(2)) and pharmacological (anti-diabetic sulfonylurea drug glimepiride) stimuli and to increase the esterification into as well as to reduce the release of fatty acids from triacylglycerol. Here microvesicles derived from (preferentially large) rat adipocytes or plasma and harboring the GPI-anchored proteins, Gce1 and CD73, were demonstrated to contain specific transcripts and microRNAs that are both transferred into and expressed in acceptor adipocytes and are involved in the upregulation of lipogenesis and cell size. The transferred transcripts were specific for fatty acid esterification (glycerol-3-phosphate acyltransferase-3, diacylglycerol acyltransferase-2), lipid droplet biogenesis (FSP27, caveolin-1) and adipokines (leptin, adiponectin). The transfer and lipogenic activity were more efficient for small rather than large acceptor adipocytes and significantly upregulated by palmitate, glimepiride and H(2)O(2). Together the data suggest that microvesicles released from large adipocytes stimulate lipid storage in small adipocytes by mediating horizontal transfer of lipogenic information which is encoded by relevant (micro)RNA and GPI-anchored protein species. Paracrine and endocrine regulation of lipid storage and, in parallel, cell size of white adipocytes by specific (micro)RNAs in GPI-anchored protein-harboring microvesicles may represent a novel target for interference with metabolic diseases, such as obesity and metabolic syndrome. (hide)
EV-METRIC
43% (82nd 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
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
9.06 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.12-1.22
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
Equal to or above 150,000 g
Pelleting: time(min)
30
Pelleting: rotor type
A110
Pelleting: adjusted k-factor
9.06
Density gradient
Only used for validation of main results
1
Lowest density fraction
10
Highest density fraction
70
Orientation
Top-down
Speed (g)
100000
Immunoaffinity capture
Selected surface protein(s)
Gce-/CD73
Characterization: Particle analysis
EV110043 1/1 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Hood JL 2011 43%

Study summary

Full title
All authors
Hood JL, San RS, Wickline SA
Journal
Cancer Res
Abstract
Exosomes are naturally occurring biological nanovesicles utilized by tumors to communicate signals t (show more...)Exosomes are naturally occurring biological nanovesicles utilized by tumors to communicate signals to local and remote cells and tissues. Melanoma exosomes can incite a proangiogenic signaling program capable of remodeling tissue matrices. In this study, we show exosome-mediated conditioning of lymph nodes and define microanatomic responses that license metastasis of melanoma cells. Homing of melanoma exosomes to sentinel lymph nodes imposes synchronized molecular signals that effect melanoma cell recruitment, extracellular matrix deposition, and vascular proliferation in the lymph nodes. Our findings highlight the pathophysiologic role and mechanisms of an exosome-mediated process of microanatomic niche preparation that facilitates lymphatic metastasis by cancer cells. (hide)
EV-METRIC
43% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Adj. k-factor
156.9 (pelleting) / 156.9 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.21
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
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
156.9
Wash: Rotor Type
70Ti
Wash: adjusted k-factor
156.9
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Characterization: Particle analysis
DLS
EV110016 1/1 Pseudomonas aeruginosa Bacteria 0.2 µm filter
0.45 µm filter
dUC
Iodixanol-DG
UF
Choi DS 2011 43%

Study summary

Full title
All authors
Choi DS, Kim DK, Choi SJ, Lee J, Choi JP, Rho S, Park SH, Kim YK, Hwang D, Gho YS
Journal
Proteomics
Abstract
Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, constitutively secretes outer mem (show more...)Pseudomonas aeruginosa, an opportunistic human bacterial pathogen, constitutively secretes outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress has revealed that OMVs are essential for pathogenesis of P. aeruginosa, their proteins have not been comprehensively analyzed so far. In this study, we identified 338 vesicular proteins with high confidence by five separate LC-MS/MS analyses. This global proteome profile provides a basis for future studies to elucidate the pathological functions of OMVs from P. aeruginosa. (hide)
EV-METRIC
43% (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
Bacteria
Focus vesicles
OMV
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + 0.45 µm filter + dUC + Iodixanol-DG + UF
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Pseudomonas aeruginosa
Sample Type
Bacteria
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
180
Density gradient
Lowest density fraction
10
Highest density fraction
50
Orientation
Bottom-up
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Wide-field
EV110028 1/3 Homo sapiens
Malassezia sympodialis
Cell culture supernatant dUC
Immunoaffinity (valid.)
Sucrose-DG (valid.)
Gehrmann U 2011 38%

Study summary

Full title
All authors
Gehrmann U, Qazi KR, Johansson C, Hultenby K, Karlsson M, Lundeberg L, Gabrielsson S, Scheynius A
Journal
PLoS One
Abstract
BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are (show more...)BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. OBJECTIVE: To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. METHODS: Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. RESULTS: We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-? responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-? responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-? but not IL-4 in undepleted PBMC. CONCLUSIONS: Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE. (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 / "Nano(-sized) 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 + Immunoaffinity (valid.) + Sucrose-DG (valid.)
Protein markers
EV: CD63/ MHC2/ MHC2
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.2
Show all info
Study aim
Function
Sample
Species
Homo sapiens / Malassezia sympodialis
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
Top-down
Speed (g)
200000
Immunoaffinity capture
Selected surface protein(s)
MHC2
Western Blot
Detected EV-associated proteins
MHC2/ MHC2
ELISA
Detected EV-associated proteins
MHC2/ MHC2
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up
EV110017 1/1 Homo sapiens Ascites Density cushion
Iodixanol-DG
Choi DS 2011 38%

Study summary

Full title
All authors
Choi DS, Park JO, Jang SC, Yoon YJ, Jung JW, Choi DY, Kim JW, Kang JS, Park J, Hwang D, Lee KH, Park SH, Kim YK, Desiderio DM, Kim KP, Gho YS
Journal
Proteomics
Abstract
The presence of malignant ascites in the peritoneal cavity is a poor prognostic indicator of low sur (show more...)The presence of malignant ascites in the peritoneal cavity is a poor prognostic indicator of low survival rate. Various cancer cells, including those of colorectal cancer (CRC), release microvesicles (exosomes) into surrounding tissues and peripheral circulation including malignant ascites. Although recent progress has revealed that microvesicles play multiple roles in tumor progression, the protein composition and the pathological function of malignant ascites-derived microvesicles are still unknown. Here, we report the first global proteomic analyses of highly purified microvesicles derived from human CRC ascites. With 1-D SDS-PAGE and nano-LC-MS/MS analyses, we identified a total of 846 microvesicular proteins from ascites of three CRC patients with high confidence; 384 proteins were identified in at least two patients. We identified proteins that might function in tumor progression via disruption of epithelial polarity, migration, invasion, tumor growth, immune modulation, and angiogenesis. Furthermore, we identified several potential diagnostic markers of CRC including colon-specific surface antigens. Our proteomic analyses will help to elucidate diverse functions of microvesicles in cancer progression and will aid in the development of novel diagnostic tools for CRC. (hide)
EV-METRIC
38% (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
Ascites
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
Density cushion + Iodixanol-DG
Protein markers
EV: Alix/ CD81/ TSG101/ Claudin3/ ICAM1/ Beta-actin
non-EV: None
Proteomics
yes
EV density (g/ml)
1.090
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Ascites
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
30
Orientation
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD81/ TSG101/ Claudin3/ ICAM1/ Beta-actin
ELISA
Detected EV-associated proteins
Claudin3/ ICAM1/ Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV110080 3/3 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
UF
Battke C 2011 38%

Study summary

Full title
All authors
Battke C, Ruiss R, Welsch U, Wimberger P, Lang S, Jochum S, Zeidler R
Journal
Cancer Immunol Immunother
Abstract
In order to grow within an immunocompetent host, tumour cells have evolved various strategies to cop (show more...)In order to grow within an immunocompetent host, tumour cells have evolved various strategies to cope with the host's immune system. These strategies include the downregulation of surface molecules and the secretion of immunosuppressive factors like IL-10 and PGE2 that impair the maturation of immune effector cells, among other mechanisms. Recently, tumour exosomes (TEX) have also been implicated in tumour-induced immune suppression as it has been shown that TEX can induce apoptosis in T lymphocytes. In this study, we extend our knowledge about immunosuppressive features of these microvesicles in that we show that TEX efficiently bind and sequester tumour-reactive antibodies and dramatically reduce their binding to tumour cells. Moreover, we demonstrate that this antibody sequestration reduces the antibody-dependent cytotoxicity by immune effector cells, which is among the most important anti-tumour reactions of the immune system and a significant activity of therapeutic antibodies. Taken together, these data point to the fact that tumour-derived exosomes interfere with the tumour-specific function of immune cells and constitute an additional mechanism how tumours escape from immune surveillance. (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
0.2 µm filter + dUC + Sucrose-DG (valid.) + UF
Adj. k-factor
25.32 (pelleting)
Protein markers
EV: HER2
non-EV: None
Proteomics
no
EV density (g/ml)
1.13-1.17
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
TL100
Pelleting: adjusted k-factor
25.32
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Filtration steps
0.22µm or 0.2µm
Western Blot
Detected EV-associated proteins
HER2
ELISA
Detected EV-associated proteins
HER2
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV110024 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Xiu F 2011 33%

Study summary

Full title
All authors
Xiu F, Côté MH, Bourgeois-Daigneault MC, Brunet A, Gauvreau MÉ, Shaw A, Thibodeau J
Journal
J Immunol
Abstract
In multivesicular bodies, HLA-DM (DM) assists the loading of antigenic peptides on classical MHC cla (show more...)In multivesicular bodies, HLA-DM (DM) assists the loading of antigenic peptides on classical MHC class II molecules such as HLA-DR. In cells expressing HLA-DO (DO), DM is redistributed from the internal vesicles to the limiting membrane of these organelles. This suggests that DO might reduce DM incorporation into exosomes, which are shed upon fusion of multivesicular bodies with the plasma membrane. To test this hypothesis, we used the 721.45 B lymphoblastoid cell line and different HeLa cell transfectants. We demonstrate that the poor recovery of DM in exosomes as compared with HLA-DR is not the mere reflection of differences in protein expression. Indeed, we found that DO contributes to the inefficient transfer of DM to exosomes. This negative regulation requires an intact di-leucine endosomal sorting motif in the cytoplasmic tail of HLA-DO?. These results demonstrate that canonical sorting signals and protein-protein interactions modulate the selection of MHC protein cargos. (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: CD9/ MHC2
non-EV: None
Proteomics
no
EV density (g/ml)
1.11-1.27
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
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
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ MHC2
ELISA
Detected EV-associated proteins
MHC2
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV110009 1/1 Homo sapiens Cell culture supernatant dUC Verweij FJ 2011 33%

Study summary

Full title
All authors
Verweij FJ, van Eijndhoven MA, Hopmans ES, Vendrig T, Wurdinger T, Cahir-McFarland E, Kieff E, Geerts D, van der Kant R, Neefjes J, Middeldorp JM, Pegtel DM
Journal
EMBO J
Abstract
The ubiquitous Epstein Barr virus (EBV) exploits human B-cell development to establish a persistent (show more...)The ubiquitous Epstein Barr virus (EBV) exploits human B-cell development to establish a persistent infection in ?90% of the world population. Constitutive activation of NF-?B by the viral oncogene latent membrane protein 1 (LMP1) has an important role in persistence, but is a risk factor for EBV-associated lymphomas. Here, we demonstrate that endogenous LMP1 escapes degradation upon accumulation within intraluminal vesicles of multivesicular endosomes and secretion via exosomes. LMP1 associates and traffics with the intracellular tetraspanin CD63 into vesicles that lack MHC II and sustain low cholesterol levels, even in 'cholesterol-trapping' conditions. The lipid-raft anchoring sequence FWLY, nor ubiquitylation of the N-terminus, controls LMP1 sorting into exosomes. Rather, C-terminal modifications that retain LMP1 in Golgi compartments preclude assembly within CD63-enriched domains and/or exosomal discharge leading to NF-?B overstimulation. Interference through shRNAs further proved the antagonizing role of CD63 in LMP1-mediated signalling. Thus, LMP1 exploits CD63-enriched microdomains to restrain downstream NF-?B activation by promoting trafficking in the endosomal-exosomal pathway. CD63 is thus a critical mediator of LMP1 function in- and outside-infected (tumour) 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
Protein markers
EV: CD63/ CD81/ HSP70/ MHC2
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 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)
60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ HSP70/ MHC2
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
MHC2
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV110067 3/3 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG
Vallhov H 2011 33%

Study summary

Full title
All authors
Vallhov H, Gutzeit C, Johansson SM, Nagy N, Paul M, Li Q, Friend S, George TC, Klein E, Scheynius A, Gabrielsson S
Journal
J Immunol
Abstract
Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes (show more...)Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV(-) B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350-CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression. (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
Protein markers
EV: CD81/ MHC2/ CD23
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.19
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
Between 50,000 g and 100,000 g
Pelleting: time(min)
60
Density gradient
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ MHC2/ CD23
ELISA
Detected EV-associated proteins
MHC2/ CD23
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EV110023 1/1 Homo sapiens Cell culture supernatant dUC Svensson KJ 2011 33%

Study summary

Full title
All authors
Svensson KJ, Kucharzewska P, Christianson HC, Sköld S, Löfstedt T, Johansson MC, Mörgelin M, Bengzon J, Ruf W, Belting M
Journal
Proc Natl Acad Sci U S A
Abstract
Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) (show more...)Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors. (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
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: CD63/ CD81/ Beta-actin
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
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ Beta-actin
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Beta-actin
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV110064 2/2 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Street JM 2011 33%

Study summary

Full title
All authors
Street JM, Birkhoff W, Menzies RI, Webb DJ, Bailey MA, Dear JW
Journal
J Physiol
Abstract
Exosomes are vesicles released following fusion of endosomes with the plasma membrane. Urine contain (show more...)Exosomes are vesicles released following fusion of endosomes with the plasma membrane. Urine contains exosomes that are released from the entire length of the nephron and change in composition with kidney disease. Exosomes can shuttle information between non-renal cells via transfer of protein and RNA. In this study murine kidney collecting duct (mCCDC11) cells were used to demonstrate that exosomes can act as a signalling mechanism between cells. First, the release of exosomes by mCCDC11 cells was confirmed by multiple approaches. Following isopynic centrifugation, exosomal proteins flotillin-1 and TSG101 were identified in fractions consistent with exosomes. Electron microscopy demonstrated structures consistent in size and shape with exosomes. Exposure of mCCDC11 cells to the synthetic vasopressin analogue, desmopressin, did not affect exosomal flotillin-1 or TSG101 but increased aquaporin 2 (AQP2) in a dose- and time-dependent manner that was highly correlated with cellular AQP2 (exosomal AQP2 vs. cellular AQP2, Pearson correlation coefficient r = 0.93). To test whether the ratio of exosomal AQP2/flotillin-1 is under physiological control in vivo, rats were treated with desmopressin. The ratio of AQP2/flotillin-1 in the urinary exosome was significantly increased. Inter-cellular signalling by exosomes was demonstrated: exosomes from desmopressin-treated cells stimulated both AQP2 expression and water transport in untreated mCCDc11 cells (water flow across cells: control exosome treatment 52.8 ± 11 ?l cm(-2); AQP2-containing exosomes 77.4 ± 4 ?l cm(-2), P = 0.05, n = 4). In summary, the amount of AQP2 in exosomes released from collecting duct cells is physiologically regulated and exosomal AQP2 closely reflects cellular expression. Exosomes can transfer functional AQP2 between cells and this represents a novel physiological mechanism for cell-to-cell communication within the kidney. (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/ TSG101/ AQP2
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.15
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 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/ AQP2
ELISA
Detected EV-associated proteins
AQP2
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV110022 1/1 Homo sapiens Urine dUC
Other-DG (valid.)
Staubach S 2011 33%

Study summary

Full title
All authors
Staubach S, Schadewaldt P, Wendel U, Nohroudi K, Hanisch FG
Journal
J Proteome Res
Abstract
A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause pro (show more...)A variety of genetic variations in the galactose-1-phosphate uridyltransferase (GALT) gene cause profound activity loss of the enzyme and acute toxic effects mediated by accumulating metabolic intermediates of galactose in newborns induced by dietary galactose. However, even on a severely galactose-restricted diet, patients develop serious long-term complications of the CNS and ovaries, which may result from damaging perturbations in cell biology caused by endogenously synthezised galactose. Under galactose stress, the cosubstrate of GALT, galactose-1-phosphate, accumulates and disturbs catabolic and anabolic pathways of the carbohydrate metabolism with potential effects on protein glycosylation and membrane localization of glycoprotein receptors, like the epidermal growth factor receptor. To address this issue in view of a cellular pathomechanism, we performed a differential semiquantitative N-glycomics study of membrane proteins. A suitable noninvasive cellular material derived from epithelial plasma membranes was found in urinary exovesicles and in the shed Tamm-Horsfall protein. By applying matrix-assisted laser ionization mass spectrometry on permethylated, PNGaseF released N-glycans, we demonstrate that GALT deficiency is associated with dramatic shifts from prevalent high-mannose-type glycans found in healthy subjects toward complex-type N-linked glycosylation in patients. These N-glycosylation shifts were observed on exosomal N-glycoproteins but not on the Tamm-Horsfall glycoprotein, which showed predominant high-mannose-type glycosylation with M6. (hide)
EV-METRIC
33% (71st 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
Exovesicles
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 + Other-DG (valid.)
Protein markers
EV: Alix/ HSP70/ Flotillin2/ MUC1
non-EV: Tamm-Horsfall glycoprotein
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Wash: volume per pellet (ml)
1.5
Density gradient
Only used for validation of main results
1
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ HSP70/ Flotillin2/ MUC1
Detected contaminants
Tamm-Horsfall glycoprotein
ELISA
Detected EV-associated proteins
Flotillin2/ MUC1
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV110011 1/3 Homo sapiens Aqueous humor dUC Stamer WD 2011 33%

Study summary

Full title
All authors
Stamer WD, Hoffman EA, Luther JM, Hachey DL, Schey KL
Journal
J Proteomics
Abstract
To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular p (show more...)To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure. (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
Aqueous humor
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
255.8 (pelleting)
Protein markers
EV: CD81/ CD9/ Flotilin1/ Annexin2/ Annexin5/ GAPDH
non-EV: None
Proteomics
no
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Aqueous humor
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW41
Pelleting: adjusted k-factor
255.8
Wash: volume per pellet (ml)
10
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ CD9/ Flotilin1/ Annexin2/ Annexin5/ GAPDH
ELISA
Detected EV-associated proteins
Annexin2/ Annexin5/ GAPDH
Characterization: Particle analysis
EV110011 2/3 Homo sapiens Urine dUC Stamer WD 2011 33%

Study summary

Full title
All authors
Stamer WD, Hoffman EA, Luther JM, Hachey DL, Schey KL
Journal
J Proteomics
Abstract
To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular p (show more...)To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure. (hide)
EV-METRIC
33% (71st 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
Adj. k-factor
255.8 (pelleting)
Protein markers
EV: CD81/ CD9/ Syntenin/ Annexin5
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW41
Pelleting: adjusted k-factor
255.8
Wash: volume per pellet (ml)
10
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ CD9/ Syntenin/ Annexin5
ELISA
Detected EV-associated proteins
Annexin5
Characterization: Particle analysis
EV110021 1/1 Homo sapiens
Rattus norvegicus/rattus
Cell culture supernatant dUC Sirois I 2011 33%

Study summary

Full title
All authors
Sirois I, Raymond MA, Brassard N, Cailhier JF, Fedjaev M, Hamelin K, Londono I, Bendayan M, Pshezhetsky AV, Hébert MJ
Journal
Cell Death Differ
Abstract
The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at (show more...)The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at sites of apoptotic cell deletion. As this paracrine pathway likely bears special importance in maladaptive intercellular communication leading to vascular remodeling, we aimed at further defining the mediators produced by apoptotic endothelial cells (EC), using comparative and functional proteomics. Apoptotic EC were found to release nanovesicles displaying ultrastructural characteristics, protein markers and functional activity that differed from apoptotic blebs. Tumor susceptibility gene 101 and translationally controlled tumor protein (TCTP) were identified in nanovesicle fractions purified from medium conditioned by apoptotic EC and absent from purified apoptotic blebs. Immunogold labeling identified TCTP on the surface of nanovesicles purified from medium conditioned by apoptotic EC and within multivesicular blebs in apoptotic EC. These nanovesicles induced an extracellular signal-regulated kinases 1/2 (ERK 1/2)-dependent antiapoptotic phenotype in vascular smooth muscle cells (VSMC), whereas apoptotic blebs did not display antiapoptotic activity on VSMC. Caspase-3 biochemical inhibition and caspase-3 RNA interference in EC submitted to a proapoptotic stimulus inhibited the release of nanovesicles. Also, TCTP siRNAs in EC attenuated the antiapoptotic activity of purified nanovesicles on VSMC. Collectively, these results identify TCTP-bearing nanovesicles as a novel component of the paracrine apoptotic program of potential importance in vascular repair. (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
255.8 (pelleting)
Protein markers
EV: TSG101
non-EV: Cell organelle protein/ Tubulin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens / Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Between 50,000 g and 100,000 g
Pelleting: time(min)
1080
Pelleting: rotor type
SW41
Pelleting: adjusted k-factor
255.8
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101
Detected contaminants
Cell organelle protein/ Tubulin
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV110008 1/1 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Lee YS 2011 33%

Study summary

Full title
All authors
Lee YS, Kim SH, Cho JA, Kim CW
Journal
Exp Mol Med
Abstract
Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes co (show more...)Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1- CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA- Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-?, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-? cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine. (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: TSG101/ MHC1/ MHC2/ Hsc70
non-EV: Cell organelle protein/ Tubulin
Proteomics
no
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
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
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101/ MHC1/ MHC2/ Hsc70
Detected contaminants
Cell organelle protein/ Tubulin
ELISA
Detected EV-associated proteins
MHC1/ MHC2/ Hsc70
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV110007 1/1 Homo sapiens Nasal fluid 0.2 µm filter
dUC
Lässer C 2011 33%

Study summary

Full title
All authors
Lässer C, O'Neil SE, Ekerljung L, Ekström K, Sjöstrand M, Lötvall J
Journal
Am J Rhinol Allergy
Abstract
BACKGROUND: Exosomes are nanovesicles of endocytic origin released by cells and present in human bod (show more...)BACKGROUND: Exosomes are nanovesicles of endocytic origin released by cells and present in human body fluids such as plasma, breast milk, and bronchoalveolar lavage fluid. These vesicles take part in communication between cells. Recently, it was shown that exosomes contain both mRNA and microRNA. This RNA can be shuttled between cells (exosomal shuttle RNA), which is a new route of communication between cells. The aim of this study was to determine whether nasal secretions harbor exosomes and furthermore, whether these exosomes contain RNA. METHODS: Human nasal lavage fluid (NLF) underwent centrifugation and filtration to discard cells and debris, followed by a final ultracentrifugation at 120,000 × g to pellet the exosomes. Exosomes were detected using electron microscopy (EM), flow cytometry, and Western blot. RNA was extracted and analyzed using a Bioanalyzer. RESULTS: Exosomes were visualized as 40-80 nm, CD63(+) vesicles using EM. Flow cytometry of exosomes using anti-major histocompatibility complex class II beads revealed exosomes positive for the tetraspanins CD9, CD63, and CD81. Western blot confirmed the presence of exosomal protein and absence of proteins from the endoplasmic reticulum (ER), because the exosomes were positive for Tsg101, but negative for the ER marker, calnexin. Bioanalyzer analysis revealed that, these exosomes contain RNA. CONCLUSION: This study shows for the first time that NLF contains exosomes and that these exosomes contain RNA. Further characterization of the exosomal RNA and proteins may provide important information about communication in the nose and potentially provide a source of biomarkers for upper airway diseases. (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
Nasal 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
Protein markers
EV: CD63/ CD81/ CD9/ TSG101
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Nasal fluid
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/ CD9/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up
EV110077 3/3 Homo sapiens Blood plasma 0.1 µm filter Grant R 2011 33%

Study summary

Full title
All authors
Grant R, Ansa-Addo E, Stratton D, Antwi-Baffour S, Jorfi S, Kholia S, Krige L, Lange S, Inal J
Journal
J Immunol Methods
Abstract
The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably (show more...)The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 ?m in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 ?m pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 ?m) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 ?M) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects. (hide)
EV-METRIC
33% (78th 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 / "Membrane(-derived) vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.1 µm filter
Protein markers
EV: CD63/ Annexin5
non-EV: None
Proteomics
no
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
Filtration steps
0.2µm > x > 0.1µm
Western Blot
Detected EV-associated proteins
Annexin5
ELISA
Detected EV-associated proteins
Annexin5
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV110040 1/1 Mus musculus Cell culture supernatant dUC
Sucrose-DG
Ghidoni R 2011 33%

Study summary

Full title
All authors
Ghidoni R, Paterlini A, Albertini V, Glionna M, Monti E, Schiaffonati L, Benussi L, Levy E, Binetti G
Journal
Neurobiol Aging
Abstract
It has recently become clear that proteins associated with neurodegenerative disorders can be select (show more...)It has recently become clear that proteins associated with neurodegenerative disorders can be selectively incorporated into intraluminal vesicles of multivesicular bodies and subsequently released within exosomes. Multiple lines of research support a neuroprotective role for cystatin C in Alzheimer's disease (AD). Herein we demonstrate that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, is also secreted by mouse primary neurons in association with exosomes. Immunoproteomic analysis using SELDI-TOF MS revealed the presence in exosomes of at least 9 different cystatin C glycoforms. Moreover, the over-expression of familial AD-associated presenilin 2 mutations (PS2 M239I and PS2 T122R) resulted in reduced levels of all cystatin C forms (native and glycosylated) and of amyloid-? precursor protein (APP) metabolites within exosomes. A better understanding of the mechanisms involved in exosomal processing and release may have important implications for the fight against AD and other neurodegenerative diseases. (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
Adj. k-factor
106.5 (pelleting)
Protein markers
EV: TSG101/ Flotillin2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
T8100
Pelleting: adjusted k-factor
106.5
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101/ Flotillin2
ELISA
Detected EV-associated proteins
Flotillin2
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV110028 2/3 Malassezia sympodialis Fungus dUC
Immunoaffinity (valid.)
Sucrose-DG (valid.)
Gehrmann U 2011 33%

Study summary

Full title
All authors
Gehrmann U, Qazi KR, Johansson C, Hultenby K, Karlsson M, Lundeberg L, Gabrielsson S, Scheynius A
Journal
PLoS One
Abstract
BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are (show more...)BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. OBJECTIVE: To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. METHODS: Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. RESULTS: We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-? responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-? responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-? but not IL-4 in undepleted PBMC. CONCLUSIONS: Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE. (hide)
EV-METRIC
33% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Fungus
Focus vesicles
exosomes / "Nano(-sized) 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 + Immunoaffinity (valid.) + Sucrose-DG (valid.)
Protein markers
EV: "MHC2/ MHC2/ M.sympodialis antigen"
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.2
Show all info
Study aim
Function
Sample
Species
Malassezia sympodialis
Sample Type
Fungus
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
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
Top-down
Speed (g)
200000
Immunoaffinity capture
Selected surface protein(s)
MHC2
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
"MHC2/ MHC2/ M.sympodialis antigen"
ELISA
Detected EV-associated proteins
"MHC2/ MHC2/ M.sympodialis antigen"
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up
EV110028 3/3 Homo sapiens Blood plasma 0.2 µm filter
dUC
Immunoaffinity (valid.)
Sucrose-DG (valid.)
Gehrmann U 2011 33%

Study summary

Full title
All authors
Gehrmann U, Qazi KR, Johansson C, Hultenby K, Karlsson M, Lundeberg L, Gabrielsson S, Scheynius A
Journal
PLoS One
Abstract
BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are (show more...)BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. OBJECTIVE: To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. METHODS: Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. RESULTS: We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-? responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-? responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-? but not IL-4 in undepleted PBMC. CONCLUSIONS: Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE. (hide)
EV-METRIC
33% (78th 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 / "Nano(-sized) 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 + Immunoaffinity (valid.) + Sucrose-DG (valid.)
Protein markers
EV: CD63/ CD81/ MHC2/ MHC2
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.18
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
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
Speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Immunoaffinity capture
Selected surface protein(s)
MHC2
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ MHC2/ MHC2
ELISA
Detected EV-associated proteins
MHC2/ MHC2
Characterization: Particle analysis
EV110039 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
UF
Gangalum RK 2011 33%

Study summary

Full title
All authors
Gangalum RK, Atanasov IC, Zhou ZH, Bhat SP
Journal
J Biol Chem
Abstract
?B-crystallin (?B) is known as an intracellular Golgi membrane-associated small heat shock protein. (show more...)?B-crystallin (?B) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of ?B has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that ?B is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of ?B in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize ?B in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100-200 nm, were characterized by the presence of flotillin-1, ?-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of ?B is predominantly apical. Using OptiPrep gradients we demonstrate that ?B resides in the DRM fraction. The secretion of ?B is inhibited by the cholesterol-depleting drug, methyl ?-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts. (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 + UF
Adj. k-factor
251.4 (pelleting) / 251.4 (washing)
Protein markers
EV: Flotilin1/ HSP70/ Alpha-enolase
non-EV: None
Proteomics
no
TEM measurements
100-200
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 100,000 g and 150,000 g
Pelleting: time(min)
960
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
251.4
Wash: volume per pellet (ml)
12
Wash: Rotor Type
SW40
Wash: adjusted k-factor
251.4
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ Alpha-enolase
ELISA
Detected EV-associated proteins
Alpha-enolase
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Wide-field
EV110081 3/3 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
Clayton A 2011 33%

Study summary

Full title
All authors
Clayton A, Al-Taei S, Webber J, Mason MD, Tabi Z
Journal
J Immunol
Abstract
Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune respo (show more...)Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 5'AMP-phosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosome-generated adenosine can trigger a cAMP response in adenosine A(2A) receptor-positive but not A(2A) receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28 cross-linking could be inhibited by exogenously added 5'AMP in a CD73-dependent manner. However, 5'AMP converted to adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated through the adenosine A(2A) receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment. (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: TSG101/ CD39/ GAPDH/ CD73
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.2
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.2
Highest density fraction
2.5
Orientation
Top-down
Speed (g)
150000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101/ CD39/ GAPDH/ CD73
ELISA
Detected EV-associated proteins
CD39/ GAPDH/ CD73
Characterization: Particle analysis
EV110014 1/1 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Chen T 2011 33%

Study summary

Full title
All authors
Chen T, Guo J, Yang M, Zhu X, Cao X
Journal
J Immunol
Abstract
Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane ve (show more...)Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane vesicles that can induce specific antitumor immunity. During investigation of the effects of hyperthermia on antitumor immune response, we found that exosomes derived from heat-stressed tumor cells (HS-TEX) could chemoattract and activate dendritic cells (DC) and T cells more potently than that by conventional tumor-derived exosomes. We show that HS-TEX contain chemokines, such as CCL2, CCL3, CCL4, CCL5, and CCL20, and the chemokine-containing HS-TEX are functionally competent in chemoattracting CD11c(+) DC and CD4(+)/CD8(+) T cells both in vitro and in vivo. Moreover, the production of chemokine-containing HS-TEX could be inhibited by ATP inhibitor, calcium chelator, and cholesterol scavenger, indicating that the mobilization of chemokines into exosomes was ATP- and calcium-dependent and via a lipid raft-dependent pathway. We consistently found that the intracellular chemokines could be enriched in lipid rafts after heat stress. Accordingly, intratumoral injection of HS-TEX could induce specific antitumor immune response more efficiently than that by tumor-derived exosomes, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice more significantly. Therefore, our results demonstrate that exosomes derived from HS-TEX represent a kind of efficient tumor vaccine and can chemoattract and activate DC and T cells, inducing more potent antitumor immune response. Release of chemokines through exosomes via lipid raft-dependent pathway may be a new method of chemokine exocytosis. (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: CD63/ HSP90/ HSP70/ Annexin2/ CD18/ CD54/ Tf-receptor/ CD80/ CD86/ CD107a/ HSP60/ HSC70/ MHC1/ MHC2
non-EV: None
Proteomics
no
EV density (g/ml)
1.12-1.17
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
Density gradient
Only used for validation of main results
1
Lowest density fraction
30
Highest density fraction
45
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ HSP90/ HSP70/ Annexin2/ CD18/ CD54/ Tf-receptor/ CD80/ CD86/ CD107a/ HSP60/ HSC70/ MHC1/ MHC2
ELISA
Detected EV-associated proteins
Annexin2/ CD18/ CD54/ Tf-receptor/ CD80/ CD86/ CD107a/ HSP60/ HSC70/ MHC1/ MHC2
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV110013 1/1 Rattus norvegicus/rattus Cell culture supernatant dUC Bakhti M 2011 33%

Study summary

Full title
All authors
Bakhti M, Winter C, Simons M
Journal
J Biol Chem
Abstract
Myelin formation is a multistep process that is controlled by a number of different extracellular fa (show more...)Myelin formation is a multistep process that is controlled by a number of different extracellular factors. During the development of the central nervous system (CNS), oligodendrocyte progenitor cells differentiate into mature oligodendrocytes that start to enwrap axons with myelin membrane sheaths after receiving the appropriate signal(s) from the axon or its microenvironment. The signals required to initiate this process are unknown. Here, we show that oligodendrocytes secrete small membrane vesicles, exosome-like vesicles, into the extracellular space that inhibit both the morphological differentiation of oligodendrocytes and myelin formation. The inhibitory effects of exosome-like vesicles were prevented by treatment with inhibitors of actomyosin contractility. Importantly, secretion of exosome-like vesicles from oligodendrocytes was dramatically reduced when cells were incubated by conditioned neuronal medium. In conclusion, our results provide new evidence for small and diffusible oligodendroglial-derived vesicular carriers within the extracellular space that have inhibitory properties on cellular growth. We propose that neurons control the secretion of autoinhibitory oligodendroglial-derived exosomes to coordinate myelin membrane biogenesis. (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/ TSG101/ Flotillin2
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ Flotillin2
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Flotillin2
Characterization: Particle analysis