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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
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Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV120010 4/6 Homo sapiens Urine Commercial Alvarez ML 2012 38%

Study summary

Full title
All authors
Alvarez ML, Khosroheidari M, Kanchi Ravi R, DiStefano JK
Journal
Kidney Int
Abstract
Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as bio (show more...)Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as biomarkers of renal dysfunction and structural injury. Currently, there is a need for more sensitive and specific biomarkers of renal injury and disease progression. Here we sought to identify the best exosome isolation methods for both proteomic analysis and RNA profiling as a first step for biomarker discovery. We used six different protocols; three were based on ultracentrifugation, one used a nanomembrane concentrator-based approach, and two utilized a commercial exosome precipitation reagent. The highest yield of exosomes was obtained using a modified exosome precipitation protocol, which also yielded the highest quantities of microRNA and mRNA and, therefore, is ideal for subsequent RNA profiling. This method is likewise suitable for downstream proteomic analyses if an ultracentrifuge is not available and/or a large number of samples are to be processed. Two of the ultracentrifugation methods, however, are better options for exosome isolation if an ultracentrifuge is available and few samples will be processed for proteomic analysis. Thus, our modified exosome precipitation method is a simple, fast, highly scalable, and effective alternative for the isolation of exosomes, and may facilitate the identification of exosomal biomarkers from urine. (hide)
EV-METRIC
38% (79th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD9/ TSG101
ELISA
Detected EV-associated proteins
Alix/ CD9/ TSG101
EV120010 5/6 Homo sapiens Urine Commercial Alvarez ML 2012 38%

Study summary

Full title
All authors
Alvarez ML, Khosroheidari M, Kanchi Ravi R, DiStefano JK
Journal
Kidney Int
Abstract
Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as bio (show more...)Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as biomarkers of renal dysfunction and structural injury. Currently, there is a need for more sensitive and specific biomarkers of renal injury and disease progression. Here we sought to identify the best exosome isolation methods for both proteomic analysis and RNA profiling as a first step for biomarker discovery. We used six different protocols; three were based on ultracentrifugation, one used a nanomembrane concentrator-based approach, and two utilized a commercial exosome precipitation reagent. The highest yield of exosomes was obtained using a modified exosome precipitation protocol, which also yielded the highest quantities of microRNA and mRNA and, therefore, is ideal for subsequent RNA profiling. This method is likewise suitable for downstream proteomic analyses if an ultracentrifuge is not available and/or a large number of samples are to be processed. Two of the ultracentrifugation methods, however, are better options for exosome isolation if an ultracentrifuge is available and few samples will be processed for proteomic analysis. Thus, our modified exosome precipitation method is a simple, fast, highly scalable, and effective alternative for the isolation of exosomes, and may facilitate the identification of exosomal biomarkers from urine. (hide)
EV-METRIC
38% (79th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 10,000 g and 50,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD9/ TSG101
ELISA
Detected EV-associated proteins
Alix/ CD9/ TSG101
EV120025 3/3 Homo sapiens Other Density cushion
SEC
Sucrose-DG (valid.)
Aalberts M 2012 38%

Study summary

Full title
All authors
Aalberts M, van Dissel-Emiliani FM, van Adrichem NP, van Wijnen M, Wauben MH, Stout TA, Stoorvogel W
Journal
Biol Reprod
Abstract
In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include (show more...)In addition to sperm cells, seminal fluid contains various small membranous vesicles. These include prostasomes, membrane vesicles secreted by prostate epithelial cells. Prostasomes have been proposed to perform a variety of functions, including modulation of (immune) cell activity within the female reproductive tract and stimulation of sperm motility and capacitation. How prostasomes mediate such diverse functions, however, remains unclear. In many studies, vesicles from the seminal plasma have been categorized collectively as a single population of prostasomes; in fact, they more likely represent a heterogeneous mixture of vesicles produced by different reproductive glands and secretory mechanisms. We here characterized membranous vesicles from seminal fluid obtained from vasectomized men, thereby excluding material from the testes or epididymides. Two distinct populations of vesicles with characteristic sizes (56 ± 13 nm vs. 105 ± 25 nm) but similar equilibrium buoyant density (?1.15 g/ml) could be separated by using the distinct rates with which they floated into sucrose gradients. Both types of vesicle resembled exosomes in terms of their buoyant density, size, and the presence of the ubiquitous exosome marker CD9. The protein GLIPR2 was found to be specifically enriched in the lumen of the smaller vesicles, while annexin A1 was uniquely associated with the surface of the larger vesicles. Prostate stem-cell antigen (PSCA), a prostate-specific protein, was present on both populations, thereby confirming their origin. PSCA was, however, absent from membrane vesicles in the seminal fluid of some donors, indicating heterogeneity of prostasome characteristics between individuals. (hide)
EV-METRIC
38% (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
Other
Focus vesicles
Prostasomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density cushion + SEC + Sucrose-DG (valid.)
Protein markers
EV: CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.12-1.20
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
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
0.4
Highest density fraction
2.5
Orientation
Top-down
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9
EV120111 1/3 Bos bovis Milk dUC
Sucrose-DG (valid.)
UF
Yamada T 2012 33%

Study summary

Full title
All authors
Yamada T, Inoshima Y, Matsuda T, Ishiguro N
Journal
J Vet Med Sci
Abstract
Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) (show more...)Four methods were evaluated for isolating exosomes from bovine milk: (1) ExoQuick precipitation, (2) ultracentrifugation with ExoQuick precipitation, (3) ultracentrifugation with density gradient centrifugation, and (4) human milk exosome isolation. Methods 1 and 4 failed due to differences between bovine and human milk. Exosomes were efficiently isolated by ultracentrifugation with either ExoQuick precipitation (method 2) or density gradient centrifugation (method 3). The highest yield of exosomes was achieved using ultracentrifugation with ExoQuick precipitation, whereas higher quality exosome isolation with intact morphological structures was achieved by ultracentrifugation with density gradient centrifugation. (hide)
EV-METRIC
33% (65th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Milk
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.) + UF
Adj. k-factor
255.8 (pelleting)
Protein markers
EV: CD9/ MFGE8
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.19
Show all info
Study aim
Technical
Sample
Species
Bos bovis
Sample Type
Milk
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Between 50,000 g and 100,000 g
Pelleting: time(min)
60
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
10
Highest density fraction
40
Orientation
Top-down
Rotor type
MLA55
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ MFGE8
ELISA
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
EM
EM-type
transmission EM
EV120048 1/1 Rattus norvegicus/rattus Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Xin H 2012 33%

Study summary

Full title
All authors
Xin H, Li Y, Buller B, Katakowski M, Zhang Y, Wang X, Shang X, Zhang ZG, Chopp M
Journal
Stem Cells
Abstract
Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of (show more...)Multipotent mesenchymal stromal cells (MSCs) have potential therapeutic benefit for the treatment of neurological diseases and injury. MSCs interact with and alter brain parenchymal cells by direct cell-cell communication and/or by indirect secretion of factors and thereby promote functional recovery. In this study, we found that MSC treatment of rats subjected to middle cerebral artery occlusion (MCAo) significantly increased microRNA 133b (miR-133b) level in the ipsilateral hemisphere. In vitro, miR-133b levels in MSCs and in their exosomes increased after MSCs were exposed to ipsilateral ischemic tissue extracts from rats subjected to MCAo. miR-133b levels were also increased in primary cultured neurons and astrocytes treated with the exosome-enriched fractions released from these MSCs. Knockdown of miR-133b in MSCs confirmed that the increased miR-133b level in astrocytes is attributed to their transfer from MSCs. Further verification of this exosome-mediated intercellular communication was performed using a cel-miR-67 luciferase reporter system and an MSC-astrocyte coculture model. Cel-miR-67 in MSCs was transferred to astrocytes via exosomes between 50 and 100 nm in diameter. Our data suggest that the cel-miR-67 released from MSCs was primarily contained in exosomes. A gap junction intercellular communication inhibitor arrested the exosomal microRNA communication by inhibiting exosome release. Cultured neurons treated with exosome-enriched fractions from MSCs exposed to 72 hours post-MCAo brain extracts significantly increased the neurite branch number and total neurite length. This study provides the first demonstration that MSCs communicate with brain parenchymal cells and may regulate neurite outgrowth by transfer of miR-133b to neural cells via exosomes. (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: Alix
non-EV: None
Proteomics
no
EV density (g/ml)
1.19-1.22
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
180
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Top-down
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120024 2/3 Homo sapiens Cell culture supernatant 0.1 µm filter
dUC
Tauro BJ 2012 33%

Study summary

Full title
All authors
Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ
Journal
Methods
Abstract
Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and p (show more...)Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling. (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
0.1 µm filter + dUC
Adj. k-factor
90.8 (pelleting)
Protein markers
EV: Alix/ HSP70/ TSG101
non-EV: None
Proteomics
yes
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
TLA45
Pelleting: adjusted k-factor
90.8
Wash: volume per pellet (ml)
1
Filtration steps
0.2µm > x > 0.1µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ HSP70/ TSG101
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120084 1/2 Aggregatibacter actinomycetemcomitans Other 0.2 µm filter
dUC
Iodixanol-DG (valid.)
Rompikuntal PK 2012 33%

Study summary

Full title
All authors
Rompikuntal PK, Thay B, Khan MK, Alanko J, Penttinen AM, Asikainen S, Wai SN, Oscarsson J
Journal
Infect Immun
Abstract
Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly (show more...)Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium. (hide)
EV-METRIC
33% (73rd 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
Other
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 + dUC + Iodixanol-DG (valid.)
Adj. k-factor
184.6 (pelleting) / 184.6 (washing)
Protein markers
EV: CdtA/ CdtB/ OmpA/ LtxA
non-EV: DNaK
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Aggregatibacter actinomycetemcomitans
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 50,000 g and 100,000 g
Pelleting: time(min)
120
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
184.6
Wash: Rotor Type
70Ti
Wash: adjusted k-factor
184.6
Density gradient
Only used for validation of main results
1
Lowest density fraction
10
Highest density fraction
45
Orientation
Bottom-up
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CdtA/ CdtB/ OmpA/ LtxA
Detected contaminants
DNaK
ELISA
Detected EV-associated proteins
CdtA/ CdtB/ OmpA/ LtxA
Characterization: Particle analysis
EM
EM-type
atomic force EM
Image type
Wide-field
EV120084 2/2 Aggregatibacter actinomycetemcomitans Other 0.2 µm filter
dUC
Iodixanol-DG (valid.)
Rompikuntal PK 2012 33%

Study summary

Full title
All authors
Rompikuntal PK, Thay B, Khan MK, Alanko J, Penttinen AM, Asikainen S, Wai SN, Oscarsson J
Journal
Infect Immun
Abstract
Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly (show more...)Aggregatibacter actinomycetemcomitans is implicated in aggressive forms of periodontitis. Similarly to several other Gram-negative species, this organism produces and excretes a cytolethal distending toxin (CDT), a genotoxin associated with cell distention, G2 cell cycle arrest, and/or apoptosis in many mammalian cell types. In this study, we have identified A. actinomycetemcomitans outer membrane vesicles (OMVs) as a vehicle for simultaneous delivery of multiple proteins, including CDT, into human cells. The OMV proteins were internalized in both HeLa cells and human gingival fibroblasts (HGF) via a mechanism of OMV fusion with lipid rafts in the plasma membrane. The active toxin unit, CdtB, was localized inside the nucleus of the intoxicated cells, whereas OmpA and proteins detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells had a perinuclear distribution. In accordance with a tight association of CdtB with OMVs, vesicles isolated from A. actinomycetemcomitans strain D7SS (serotype a), in contrast to OMVs from a D7SS cdtABC mutant, induced a cytolethal distending effect on HeLa and HGF cells, indicating that OMV-associated CDT was biologically active. Association of CDT with OMVs was also observed in A. actinomycetemcomitans isolates belonging to serotypes b and c, indicating that OMV-mediated release of CDT may be conserved in A. actinomycetemcomitans. Although the role of A. actinomycetemcomitans OMVs in periodontal disease has not yet been elucidated, our present data suggest that OMVs could deliver biologically active CDT and additional virulence factors into susceptible cells of the periodontium. (hide)
EV-METRIC
33% (73rd 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
Other
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 + dUC + Iodixanol-DG (valid.)
Adj. k-factor
184.6 (pelleting) / 184.6 (washing)
Protein markers
EV: CdtA/ CdtB/ OmpA/ LtxA
non-EV: DNaK
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Aggregatibacter actinomycetemcomitans
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 50,000 g and 100,000 g
Pelleting: time(min)
120
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
184.6
Wash: Rotor Type
70Ti
Wash: adjusted k-factor
184.6
Density gradient
Only used for validation of main results
1
Lowest density fraction
10
Highest density fraction
45
Orientation
Bottom-up
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CdtA/ CdtB/ OmpA/ LtxA
Detected contaminants
DNaK
ELISA
Detected EV-associated proteins
CdtA/ CdtB/ OmpA/ LtxA
Characterization: Particle analysis
EM
EM-type
atomic force EM
Image type
Wide-field
EV120079 1/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
UF
Nabhan JF 2012 33%

Study summary

Full title
All authors
Nabhan JF, Hu R, Oh RS, Cohen SN, Lu Q
Journal
Proc Natl Acad Sci U S A
Abstract
Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The l (show more...)Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses. (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
0.2 µm filter + dUC + UF
Adj. k-factor
213.2 (pelleting)
Protein markers
EV: CD63/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
SW41
Pelleting: adjusted k-factor
213.2
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ TSG101
Characterization: Particle analysis
EV120079 2/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
UF
Nabhan JF 2012 33%

Study summary

Full title
All authors
Nabhan JF, Hu R, Oh RS, Cohen SN, Lu Q
Journal
Proc Natl Acad Sci U S A
Abstract
Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The l (show more...)Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses. (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
0.2 µm filter + dUC + UF
Adj. k-factor
54.74 (pelleting)
Protein markers
EV: CD63/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
TLA110
Pelleting: adjusted k-factor
54.74
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ TSG101
Characterization: Particle analysis
EV120035 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Lv LH 2012 33%

Study summary

Full title
All authors
Lv LH, Wan YL, Lin Y, Zhang W, Yang M, Li GL, Lin HM, Shang CZ, Chen YJ, Min J
Journal
J Biol Chem
Abstract
Failure of immune surveillance related to inadequate host antitumor immune responses has been sugges (show more...)Failure of immune surveillance related to inadequate host antitumor immune responses has been suggested as a possible cause of the high incidence of recurrence and poor overall survival outcome of hepatocellular carcinoma. The stress-induced heat shock proteins (HSPs) are known to act as endogenous danger signals that can improve tumor immunogenicity and induce natural killer (NK) cell responses. Exosome is a novel secretory pathway for HSPs. In our experiments, the immune regulatory effect of the HSP-bearing exosomes secreted by human hepatocellular carcinoma cells under stress conditions on NK cells was studied. ELISA results showed that the production of HSP60, HSP70, and HSP90 was up-regulated in both cell lines in a stress-specific manner. After exposure to hepatocellular carcinoma cell-resistant or sensitive anticancer drugs (hereafter referred to as resistant or sensitive anticancer drug), the membrane microvesicles were actively released by hepatocellular carcinoma cells, differing in their ability to present HSPs on the cell surface, which were characterized as exosomes. Acting as a decoy, the HSP-bearing exosomes efficiently stimulated NK cell cytotoxicity and granzyme B production, up-regulated the expression of inhibitory receptor CD94, and down-regulated the expression of activating receptors CD69, NKG2D, and NKp44. Notably, resistant anticancer drugs enhanced exosome release and generated more exosome-carried HSPs, which augmented the activation of the cytotoxic response. In summary, our findings demonstrated that exosomes derived from resistant anticancer drug-treated HepG2 cells conferred superior immunogenicity in inducing HSP-specific NK cell responses, which provided a clue for finding an efficient vaccine for hepatocellular carcinoma immunotherapy. (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
Adj. k-factor
232.5 (pelleting) / 232.5 (washing)
Protein markers
EV: CD63/ HSP90/ HSP70/ HSP60/ GAPDH/ AChE
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 ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
180
Pelleting: rotor type
SW41
Pelleting: adjusted k-factor
232.5
Wash: Rotor Type
SW41
Wash: adjusted k-factor
232.5
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ HSP90/ HSP70/ HSP60/ GAPDH/ AChE
ELISA
Detected EV-associated proteins
HSP60/ GAPDH/ AChE
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV120071 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Lee HD 2012 33%

Study summary

Full title
All authors
Lee HD, Koo BH, Kim YH, Jeon OH, Kim DS
Journal
FASEB J
Abstract
A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RG (show more...)A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin ?v?3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity. (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.)
Adj. k-factor
138.6 (pelleting)
Protein markers
EV: CD9/ TSG101/ GAPDH/ ADAM15
non-EV: None
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 ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW55
Pelleting: adjusted k-factor
138.6
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ TSG101/ GAPDH/ ADAM15
ELISA
Detected EV-associated proteins
GAPDH/ ADAM15
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV120070 1/1 Mus musculus Cell culture supernatant dUC
Iodixanol-DG
Lee EY 2012 33%

Study summary

Full title
All authors
Lee EY, Park KS, Yoon YJ, Lee J, Moon HG, Jang SC, Choi KH, Kim YK, Gho YS
Journal
PLoS One
Abstract
Cancer vaccines with optimal tumor-associated antigens show promise for anti-tumor immunotherapy. Re (show more...)Cancer vaccines with optimal tumor-associated antigens show promise for anti-tumor immunotherapy. Recently, nano-sized vesicles, such as exosomes derived from tumors, were suggested as potential antigen candidates, although the total yield of exosomes is not sufficient for clinical applications. In the present study, we developed a new vaccine strategy based on nano-sized vesicles derived from primary autologous tumors. Through homogenization and sonication of tumor tissues, we achieved high yields of vesicle-bound antigens. These nanovesicles were enriched with antigenic membrane targets but lacked nuclear autoantigens. Furthermore, these nanovesicles together with adjuvant activated dendritic cells in vitro, and induced effective anti-tumor immune responses in both primary and metastatic melanoma mouse models. Therefore, autologous tumor-derived nanovesicles may represent a novel source of antigens with high-level immunogenicity for use in acellular vaccines without compromising safety. Our strategy is cost-effective and can be applied to patient-specific cancer therapeutic vaccination. (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
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 + Iodixanol-DG
Protein markers
EV: Tyrosinase/ Melan-A/ Histone
non-EV: None
Proteomics
no
EV density (g/ml)
1.087
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Density gradient
Lowest density fraction
5
Highest density fraction
20
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Tyrosinase/ Melan-A/ Histone
ELISA
Detected EV-associated proteins
Tyrosinase/ Melan-A/ Histone
Characterization: Particle analysis
EV120033 1/1 Drosophila melanogaster Other Density cushion
dUC
Koles K 2012 33%

Study summary

Full title
All authors
Koles K, Nunnari J, Korkut C, Barria R, Brewer C, Li Y, Leszyk J, Zhang B, Budnik V
Journal
J Biol Chem
Abstract
Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanis (show more...)Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release. (hide)
EV-METRIC
33% (73rd 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
Other
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 + dUC
Protein markers
EV: Alix/ Lbm
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Drosophila melanogaster
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Lbm
ELISA
Detected EV-associated proteins
Lbm
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV120021 1/2 Homo sapiens Cell culture supernatant dUC King HW 2012 33%

Study summary

Full title
All authors
King HW, Michael MZ, Gleadle JM
Journal
BMC Cancer
Abstract
BACKGROUND: Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signall (show more...)BACKGROUND: Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling. METHODS: Breast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant. RESULTS: Exposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1? siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions. CONCLUSIONS: These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1?. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD63/ Flotilin1/ TSG101/ Cofilin
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 ultra centrifugation
Differential UC: filtering 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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ TSG101/ Cofilin
ELISA
Detected EV-associated proteins
Cofilin
Characterization: Particle analysis
NTA
EM
EM-type
immune EM
Image type
Close-up
EV120012 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Chiba M 2012 33%

Study summary

Full title
All authors
Chiba M, Kimura M, Asari S
Journal
Oncol Rep
Abstract
Exosomes are microvesicles that are released from various cells into the extracellular space. It has (show more...)Exosomes are microvesicles that are released from various cells into the extracellular space. It has been reported that the components within exosomes vary according to the type of secreted cell. In the present study, we investigated the tetraspanin family proteins CD63, CD9 and CD81 as useful collection markers of exosomes derived from the three colorectal cancer (CRC) cell lines HCT-15, SW480 and WiDr. In addition, we aimed to detect the mRNAs, microRNAs and natural antisense RNAs within the exosomes secreted from the three CRC cell lines. Furthermore, we examined whether exosomes containing their RNAs were transferred into the hepatoma cell line HepG2 and lung cancer cell line A549. CD81 was detected in exosomes secreted from the three CRC cell lines. This result indicates that CD81 can be a collection marker of exosomes derived from the three CRC cell lines. When the RNA species within exosomes derived from the three CRC cell lines were examined, the mRNAs of housekeeping genes such as ACTB and GAPDH, the microRNAs such as miR-21, miR-192 and miR-221, and the natural antisense RNAs of LRRC24, MDM2 and CDKN1A genes, were detected. We discovered their natural antisense RNAs within exosomes for the first time in the present study. Furthermore, PKH67-labeled exosomes derived from the CRC cell lines were taken up into HepG2 and A549 cells. These findings indicate that the intracellular RNAs enclosed within exosomes are secreted to the outside, and exosomes derived from the CRC cell lines are transferred into HepG2 and A549 cells. In conclusion, we reveal that exosomes derived from the CRC cell lines contain mRNAs, microRNAs and natural antisense RNAs, and can be delivered into HepG2 and A549 cells. These findings indicate that exosomal RNAs can shuttle between cells, and may be involved in the regulation of gene expression in recipient cells. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Protein markers
EV: CD63/ CD81/ CD9
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 ultra centrifugation
Differential UC: filtering 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)
180
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
EV120030 1/1 Homo sapiens Urine dUC Chen CL 2012 33%

Study summary

Full title
All authors
Chen CL, Lai YF, Tang P, Chien KY, Yu JS, Tsai CH, Chen HW, Wu CC, Chung T, Hsu CW, Chen CD, Chang YS, Chang PL, Chen YT
Journal
J Proteome Res
Abstract
Bladder cancer is a common urologic cancer whose incidence continues to rise annually. Urinary micro (show more...)Bladder cancer is a common urologic cancer whose incidence continues to rise annually. Urinary microparticles are an attractive material for noninvasive bladder cancer biomarker discovery. In this study, we applied isotopic dimethylation labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to discover bladder cancer biomarkers in urinary microparticles isolated from hernia (control) and bladder cancer patients. This approach identified 2964 proteins based on more than two distinct peptides, of which 2058 had not previously been reported as constituents of human urine exosomes/microparticles. A total of 107 differentially expressed proteins were identified as candidate biomarkers. Differences in the concentrations of 29 proteins (41 signature peptides) were precisely quantified by LC-MRM/MS in 48 urine samples of bladder cancer, hernia, and urinary tract infection/hematuria. Concentrations of 24 proteins changed significantly (p<0.05) between bladder cancer (n=28) and hernia (n=12), with area-under-the-curve values ranging from 0.702 to 0.896. Finally, we quantified tumor-associated calcium-signal transducer 2 (TACSTD2) in raw urine specimens (n=221) using a commercial ELISA and confirmed its potential value for diagnosis of bladder cancer. Our study reveals a strong association of TACSTD2 with bladder cancer and highlights the potential of human urinary microparticles in the noninvasive diagnosis of bladder cancer. (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
microparticles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD9/ TSG101
non-EV: None
Proteomics
yes
TEM measurements
30-100
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Wash: volume per pellet (ml)
5
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ TSG101
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120020 1/2 Mus musculus Cell culture supernatant dUC Bobrie A 2012 33%

Study summary

Full title
All authors
Bobrie A, Colombo M, Krumeich S, Raposo G, Théry C
Journal
J Extracell Vesicles
Abstract
Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosom (show more...)Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome-derived vesicles. We propose that the most commonly used protocols for exosome preparations co-purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles. (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/ CD63/ CD9/ HSP70/ TSG101/ MFGE8
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD9/ HSP70/ TSG101/ MFGE8
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Wide-field
EV120020 2/2 Mus musculus Cell culture supernatant dUC
Sucrose-DG
Bobrie A 2012 33%

Study summary

Full title
All authors
Bobrie A, Colombo M, Krumeich S, Raposo G, Théry C
Journal
J Extracell Vesicles
Abstract
Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosom (show more...)Exosomes are extracellular vesicles of 50 to 100 nm in diameter, released by many cell types. Exosomes are formed inside the cell in intracellular endosomal compartments and are secreted upon fusion of these compartments with the plasma membrane. Cells also secrete other types of membrane vesicles, for instance, by outward budding from the plasma membrane, and although some of them clearly differ from exosomes by their structural features (larger size), others are possibly more difficult to separate. Here, using Rab27a inhibition to modulate exosome secretion, we show the existence of at least 2 distinct populations of vesicles after purification by classical ultracentrifugation from mouse tumor cell conditioned medium. Rab27a inhibition lead to decreased vesicular secretion of some conventional markers of exosomes (CD63, Tsg101, Alix and Hsc70) but did not affect secretion of others (CD9 and Mfge8). By electron microscopy, CD9 was observed on vesicles of various sizes, ranging from 30 nm to more than 150 nm in diameter. Flotation onto sucrose gradients showed different proportions of CD63, CD9 and Mfge8 not only in fractions of densities classically described for exosomes (around 1.15 g/ml) but also in fractions of densities over 1.20 g/ml, indicating the presence of heterogenous vesicle populations. CD9 and Mfge8 were also found in large vesicles pelleted at low speed and can thus not be considered as specific components of endosome-derived vesicles. We propose that the most commonly used protocols for exosome preparations co-purify vesicles from endosomal and other origins, possibly the plasma membrane. Future work will be required to improve techniques for accurate purification and characterization of the different populations of extracellular vesicles. (hide)
EV-METRIC
33% (75th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Protein markers
EV: CD63/ CD9/ MFGE8
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.14;1.26-1.29
Show all info
Study aim
Technical
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
0.4
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9/ MFGE8
ELISA
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
EV120010 2/6 Homo sapiens Urine Density cushion
dUC
Alvarez ML 2012 33%

Study summary

Full title
All authors
Alvarez ML, Khosroheidari M, Kanchi Ravi R, DiStefano JK
Journal
Kidney Int
Abstract
Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as bio (show more...)Urinary exosomes are 40-100 nm vesicles containing protein, mRNA, and microRNA that may serve as biomarkers of renal dysfunction and structural injury. Currently, there is a need for more sensitive and specific biomarkers of renal injury and disease progression. Here we sought to identify the best exosome isolation methods for both proteomic analysis and RNA profiling as a first step for biomarker discovery. We used six different protocols; three were based on ultracentrifugation, one used a nanomembrane concentrator-based approach, and two utilized a commercial exosome precipitation reagent. The highest yield of exosomes was obtained using a modified exosome precipitation protocol, which also yielded the highest quantities of microRNA and mRNA and, therefore, is ideal for subsequent RNA profiling. This method is likewise suitable for downstream proteomic analyses if an ultracentrifuge is not available and/or a large number of samples are to be processed. Two of the ultracentrifugation methods, however, are better options for exosome isolation if an ultracentrifuge is available and few samples will be processed for proteomic analysis. Thus, our modified exosome precipitation method is a simple, fast, highly scalable, and effective alternative for the isolation of exosomes, and may facilitate the identification of exosomal biomarkers from urine. (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
Density cushion + dUC
Protein markers
EV: Alix/ CD9/ TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD9/ TSG101
ELISA
Detected EV-associated proteins
Alix/ CD9/ TSG101
Characterization: Particle analysis
EV120104 1/1 Homo sapiens Other 0.8 µm filter
dUC
György B 2012 29%

Study summary

Full title
All authors
György B, Szabó TG, Turiák L, Wright M, Herczeg P, Lédeczi Z, Kittel A, Polgár A, Tóth K, Dérfalvi B, Zelenák G, Böröcz I, Carr B, Nagy G, Vékey K, Gay S, Falus A, Buzás EI
Journal
PLoS One
Abstract
INTRODUCTION: Microvesicles (MVs), earlier referred to as microparticles, represent a major type of (show more...)INTRODUCTION: Microvesicles (MVs), earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF) samples of patients with osteoarthritis (OA), rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA). To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM), Nanoparticle Tracking Analysis (NTA) and mass spectrometry (MS). For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+) and CD8(+) T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections). In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction). CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures. (hide)
EV-METRIC
29% (66th 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
Other
Focus vesicles
microvesicles / microparticles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.8 µm filter + dUC
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Filtration steps
> 0.45 µm,
Characterization: Protein analysis
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
NTA
Particle analysis: flow cytometry
EM
EM-type
transmission EM
Image type
Wide-field
EV120170 1/1 Pseudomonas aeruginosa Other 0.45 µm filter
dUC
Wessel AK 2012 29%

Study summary

Full title
All authors
Wessel AK, Liew J, Kwon T, Marcotte EM, Whiteley M
Journal
J Bacteriol
Abstract
Gram-negative bacteria produce outer membrane vesicles (OMVs) that package and deliver proteins, sma (show more...)Gram-negative bacteria produce outer membrane vesicles (OMVs) that package and deliver proteins, small molecules, and DNA to prokaryotic and eukaryotic cells. The molecular details of OMV biogenesis have not been fully elucidated, but peptidoglycan-associated outer membrane proteins that tether the outer membrane to the underlying peptidoglycan have been shown to be critical for OMV formation in multiple Enterobacteriaceae. In this study, we demonstrate that the peptidoglycan-associated outer membrane proteins OprF and OprI, but not OprL, impact production of OMVs by the opportunistic pathogen Pseudomonas aeruginosa. Interestingly, OprF does not appear to be important for tethering the outer membrane to peptidoglycan but instead impacts OMV formation through modulation of the levels of the Pseudomonas quinolone signal (PQS), a quorum signal previously shown by our laboratory to be critical for OMV formation. Thus, the mechanism by which OprF impacts OMV formation is distinct from that for other peptidoglycan-associated outer membrane proteins, including OprI. (hide)
EV-METRIC
29% (66th 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
Other
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
0.45 µm filter + dUC
Adj. k-factor
59.21 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Pseudomonas aeruginosa
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
59.21
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Characterization: Particle analysis
EV120095 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Wahlgren J 2012 29%

Study summary

Full title
All authors
Wahlgren J, Karlson Tde L, Glader P, Telemo E, Valadi H
Journal
PLoS One
Abstract
It has previously been shown that nano-meter sized vesicles (30-100 nm), exosomes, secreted by antig (show more...)It has previously been shown that nano-meter sized vesicles (30-100 nm), exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3? T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3? T cells have on resting CD3? T cells by studying T cell proliferation, cytokine production and by performing T cell and exosome phenotype characterization. Human exosomes were generated in vitro following CD3? T cell stimulation with anti-CD28, anti-CD3 and IL-2. Our results show that exosomes purified from stimulated CD3? T cells together with IL-2 were able to generate proliferation in autologous resting CD3? T cells. The CD3? T cells stimulated with exosomes together with IL-2 had a higher proportion of CD8? T cells and had a different cytokine profile compared to controls. These results indicate that activated CD3? T cells communicate with resting autologous T cells via exosomes. (hide)
EV-METRIC
29% (68th 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
130.7 (pelleting)
Protein markers
EV: CD63/ CD81/ CD9/ MHC1/ MHC2/ ICAM1
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 ultra centrifugation
Differential UC: filtering 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
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
130.7
Filtration steps
0.22µm or 0.2µm
Western Blot
Detected EV-associated proteins
MHC1/ MHC2/ ICAM1
ELISA
Detected EV-associated proteins
MHC1/ MHC2/ ICAM1
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
DLS
Particle analysis: flow cytometry
EV120090 3/3 Homo sapiens Other 0.2 µm filter
Density cushion
dUC
Sucrose-DG
Tolosa JM 2012 29%

Study summary

Full title
All authors
Tolosa JM, Schjenken JE, Clifton VL, Vargas A, Barbeau B, Lowry P, Maiti K, Smith R
Journal
Placenta
Abstract
OBJECTIVES: To examine whether syncytin-1 has immune regulatory functions and is carried by human pl (show more...)OBJECTIVES: To examine whether syncytin-1 has immune regulatory functions and is carried by human placental exosomes. Further, to examine whether corticotropin-releasing hormone (CRH) can induce the production of syncytin-1. STUDY DESIGN: Human placental exosomes were isolated from placental explant, primary trophoblast and BeWo cell cultures. The presence of exosomes was confirmed by transmission electron microscopy and western blotting. Exosomal protein was probed with 3 separate antibodies targeting syncytin-1. Syncytin-1 immunosuppression was tested, using either a syncytin-1 recombinant ectodomain protein or a synthetic peptide with the human syncytin-1 immunosuppressive domain sequence, in an in vitro human blood culture system immune challenged with LPS or PHA. The inhibition of cytokine production by syncytin-1 was determined by ELISA of TNF-?, IFN-? and CXCL10. BeWo cells were stimulated with CRH or vehicle for 24 h. mRNA and Protein was extracted from the cells for real-time PCR and western blotting analysis while exosomes were extracted from conditioned media for analysis by western blotting. RESULTS: Protein expression of syncytin-1 was detected in exosomes isolated from placental explants, primary trophoblast and BeWo cell cultures. Syncytin-1 recombinant ectodomain was also shown to inhibit the production of the Th1 cytokines TNF-? and IFN-? as well as the chemokine, CXCL10 in human blood cells. Finally, this study showed that syncytin-1 can be stimulated by CRH. CONCLUSIONS: The presence of syncytin-1 in placental exosomes provides a mechanism for syncytin-1 to reach and interact with target cells of the maternal immune system and represents a novel mechanism of endogenous retroviral mediated immunosuppression that may be relevant for maternal immune tolerance. (hide)
EV-METRIC
29% (66th 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
Other
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Density cushion + dUC + Sucrose-DG
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.16-1.19
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
Density gradient
Lowest density fraction
20
Highest density fraction
60
Orientation
Top-down
Speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120088 1/2 Dictyostelium discoideum Cell culture supernatant dUC Tatischeff I 2012 29%

Study summary

Full title
All authors
Tatischeff I, Larquet E, Falcón-Pérez JM, Turpin PY, Kruglik SG
Journal
J Extracell Vesicles
Abstract
The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-elec (show more...)The joint use of 3 complementary techniques, namely, nanoparticle tracking analysis (NTA), cryo-electron microscopy (Cryo-EM) and Raman tweezers microspectroscopy (RTM), is proposed for a rapid characterisation of extracellular vesicles (EVs) of various origins. NTA is valuable for studying the size distribution and concentration, Cryo-EM is outstanding for the morphological characterisation, including observation of vesicle heterogeneity, while RTM provides the global chemical composition without using any exogenous label. The capabilities of this approach are evaluated on the example of cell-derived vesicles of Dictyostelium discoideum, a convenient general model for eukaryotic EVs. At least 2 separate species differing in chemical composition (relative amounts of DNA, lipids and proteins, presence of carotenoids) were found for each of the 2 physiological states of this non-pathogenic microorganism, that is, cell growth and starvation-induced aggregation. These findings demonstrate the specific potency of RTM. In addition, the first Raman spectra of human urinary exosomes are reported, presumably constituting the primary step towards Raman characterisation of EVs for the purpose of human diseases diagnoses. (hide)
EV-METRIC
29% (68th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Dictyostelium discoideum
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
30
Characterization: Particle analysis
NTA
EM
EM-type
cryo EM
Image type
Close-up, Wide-field
EV120087 1/3 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Singh PP 2012 29%

Study summary

Full title
All authors
Singh PP, Smith VL, Karakousis PC, Schorey JS
Journal
J Immunol
Abstract
More than 2 billion people are infected with Mycobacterium. tuberculosis; however, only 5-10% of tho (show more...)More than 2 billion people are infected with Mycobacterium. tuberculosis; however, only 5-10% of those infected will develop active disease. Recent data suggest that containment is controlled locally at the level of the granuloma and that granuloma architecture may differ even within a single infected individual. Formation of a granuloma likely requires exposure to mycobacterial components released from infected macrophages, but the mechanism of their release is still unclear. We hypothesize that exosomes, which are small membrane vesicles containing mycobacterial components released from infected macrophages, could promote cellular recruitment during granuloma formation. In support of this hypothesis, we found that C57BL/6 mouse-derived bone marrow macrophages treated with exosomes released from M. tuberculosis-infected RAW264.7 cells secrete significant levels of chemokines and can induce migration of CFSE-labeled macrophages and splenocytes. Exosomes isolated from the serum of M. bovis bacillus Calmette-Guérin-infected mice could also stimulate macrophage production of chemokines and cytokines ex vivo, but the level and type differed during the course of a 60-d infection. Of interest, the exosome concentration in serum correlated strongly with mouse bacterial load, suggesting some role in immune regulation. Finally, hollow fiber-based experiments indicated that macrophages treated with exosomes released from M. tuberculosis-infected cells could promote macrophage recruitment in vivo. Exosomes injected intranasally could also recruit CD11b(+) cells into the lung. Overall, our study suggests that exosomes may play an important role in recruiting and regulating host cells during an M. tuberculosis infection. (hide)
EV-METRIC
29% (68th 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: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.13-1.18
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
Orientation
Bottom-up
Speed (g)
100000
Pelleting-wash: volume per pellet (mL)
3
Characterization: Particle analysis
NTA
EV120157 1/1 Bartonella henselae Other 0.2 µm filter
dUC
Roden JA 2012 29%

Study summary

Full title
All authors
Roden JA, Wells DH, Chomel BB, Kasten RW, Koehler JE
Journal
Infect Immun
Abstract
Bartonella species are gram-negative, emerging bacterial pathogens found in two distinct environment (show more...)Bartonella species are gram-negative, emerging bacterial pathogens found in two distinct environments. In the gut of the obligately hematophagous arthropod vector, bartonellae are exposed to concentrations of heme that are toxic to other bacteria. In the bloodstream of the mammalian host, access to heme and iron is severely restricted. Bartonellae have unusually high requirements for heme, which is their only utilizable source of iron. Although heme is essential for Bartonella survival, little is known about genes involved in heme acquisition and detoxification. We developed a strategy for high-efficiency transposon mutagenesis to screen for genes in B. henselae heme binding and uptake pathways. We identified a B. henselae transposon mutant that constitutively expresses the hemin binding protein C (hbpC) gene. In the wild-type strain, transcription of B. henselae hbpC was upregulated at arthropod temperature (28°C), compared to mammalian temperature (37°C). In the mutant strain, temperature-dependent regulation was absent. We demonstrated that HbpC binds hemin and localizes to the B. henselae outer membrane and outer membrane vesicles. Overexpression of hbpC in B. henselae increased resistance to heme toxicity, implicating HbpC in protection of B. henselae from the toxic levels of heme present in the gut of the arthropod vector. Experimental inoculation of cats with B. henselae strains demonstrated that both constitutive expression and deletion of hbpC affect the ability of B. henselae to infect the cat host. Modulation of hbpC expression appears to be a strategy employed by B. henselae to survive in the arthropod vector and the mammalian host. (hide)
EV-METRIC
29% (66th 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
Other
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 + dUC
Adj. k-factor
139.8 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Other/Function of a bacterial protein in infection
Sample
Species
Bartonella henselae
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
45Ti
Pelleting: adjusted k-factor
139.8
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120038 1/2 Homo sapiens Other 0.2 µm filter
dUC
Perez-Gonzalez R 2012 29%

Study summary

Full title
All authors
Perez-Gonzalez R, Gauthier SA, Kumar A, Levy E
Journal
J Biol Chem
Abstract
In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-? precur (show more...)In vitro studies have shown that neuronal cell cultures secrete exosomes containing amyloid-? precursor protein (APP) and the APP-processing products, C-terminal fragments (CTFs) and amyloid-? (A?). We investigated the secretion of full-length APP (flAPP) and APP CTFs via the exosome secretory pathway in vivo. To this end, we developed a novel protocol designed to isolate exosomes secreted into mouse brain extracellular space. Exosomes with typical morphology were isolated from freshly removed mouse brains and from frozen mouse and human brain tissues, demonstrating that exosomes can be isolated from post-mortem tissue frozen for long periods of time. flAPP, APP CTFs, and enzymes that cleave both flAPP and APP CTFs were identified in brain exosomes. Although higher levels of both flAPP and APP CTFs were observed in exosomes isolated from the brains of transgenic mice overexpressing human APP (Tg2576) compared with wild-type control mice, there was no difference in the number of secreted brain exosomes. These data indicate that the levels of flAPP and APP CTFs associated with exosomes mirror the cellular levels of flAPP and APP CTFs. Interestingly, exosomes isolated from the brains of both Tg2576 and wild-type mice are enriched with APP CTFs relative to flAPP. Thus, we hypothesize that the exosome secretory pathway plays a pleiotropic role in the brain: exosome secretion is beneficial to the cell, acting as a specific releasing system of neurotoxic APP CTFs and A?, but the secretion of exosomes enriched with APP CTFs, neurotoxic proteins that are also a source of secreted A?, is harmful to the brain. (hide)
EV-METRIC
29% (66th 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
Other
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: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Homo sapiens
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
Wash: volume per pellet (ml)
60
Filtration steps
0.22µm or 0.2µm
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV120148 1/1 Mus musculus Cell culture supernatant 0.45 µm filter
dUC
UF
Munich S 2012 29%

Study summary

Full title
All authors
Munich S, Sobo-Vujanovic A, Buchser WJ, Beer-Stolz D, Vujanovic NL
Journal
Oncoimmunology
Abstract
Autocrine and paracrine cell communication can be conveyed by multiple mediators, including membrane (show more...)Autocrine and paracrine cell communication can be conveyed by multiple mediators, including membrane-associate proteins, secreted proteins and exosomes. Exosomes are 30-100 nm endosome-derived vesicles consisting in cytosolic material surrounded by a lipid bilayer containing transmembrane proteins. We have previously shown that dendritic cells (DCs) express on their surface multiple TNF superfamily ligands (TNFSFLs), by which they can induce the apoptotic demise of tumor cells as well as the activation of natural killer (NK) cells. In the present study, we demonstrate that, similar to DCs, DC-derived exosomes (DCex) express on their surface TNF, FasL and TRAIL, by which they can trigger caspase activation and apoptosis in tumor cells. We also show that DCex activate NK cells and stimulate them to secrete interferon? (IFN?) upon the interaction of DCex TNF with NK-cell TNF receptors. These data demonstrate that DCex can mediate essential innate immune functions that were previously ascribed to DCs. (hide)
EV-METRIC
29% (68th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + dUC + UF
Adj. k-factor
126 (pelleting)
Protein markers
EV: MHC1/ MHC2
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
90Ti
Pelleting: adjusted k-factor
126.0
Filtration steps
0.45µm > x > 0.22µm,
Western Blot
Detected EV-associated proteins
MHC1/ MHC2
ELISA
Detected EV-associated proteins
MHC1/ MHC2
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
transmission EM
Image type
Wide-field
EV120077 1/2 Mus musculus Cell culture supernatant 0.2 µm filter
dUC
Marton A 2012 29%

Study summary

Full title
All authors
Marton A, Vizler C, Kusz E, Temesfoi V, Szathmary Z, Nagy K, Szegletes Z, Varo G, Siklos L, Katona RL, Tubak V, Howard OM, Duda E, Minarovits J, Nagy K, Buzas K
Journal
Immunol Lett
Abstract
To clarify controversies in the literature of the field, we have purified and characterized B16F1 me (show more...)To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-?B activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-?B activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms. (hide)
EV-METRIC
29% (68th 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
104.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
104.6
Filtration steps
0.22µm or 0.2µm
Characterization: Particle analysis
EM
EM-type
transmission EM/ atomic force EM
Image type
Close-up, Wide-field
EV120077 2/2 Mus musculus Cell culture supernatant 0.2 µm filter
dUC
Marton A 2012 29%

Study summary

Full title
All authors
Marton A, Vizler C, Kusz E, Temesfoi V, Szathmary Z, Nagy K, Szegletes Z, Varo G, Siklos L, Katona RL, Tubak V, Howard OM, Duda E, Minarovits J, Nagy K, Buzas K
Journal
Immunol Lett
Abstract
To clarify controversies in the literature of the field, we have purified and characterized B16F1 me (show more...)To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-?B activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-?B activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms. (hide)
EV-METRIC
29% (68th 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
81.62 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
T1270
Pelleting: adjusted k-factor
81.62
Filtration steps
0.22µm or 0.2µm
Characterization: Particle analysis
EM
EM-type
transmission EM/ atomic force EM
Image type
Close-up, Wide-field
EV120063 1/1 Homo sapiens Cell culture supernatant dUC Hergenreider E 2012 29%

Study summary

Full title
All authors
Hergenreider E, Heydt S, Tréguer K, Boettger T, Horrevoets AJ, Zeiher AM, Scheffer MP, Frangakis AS, Yin X, Mayr M, Braun T, Urbich C, Boon RA, Dimmeler S
Journal
Nat Cell Biol
Abstract
The shear-responsive transcription factor Krüppel-like factor 2 (KLF2) is a critical regulator of e (show more...)The shear-responsive transcription factor Krüppel-like factor 2 (KLF2) is a critical regulator of endothelial gene expression patterns induced by atheroprotective flow. As microRNAs (miRNAs) post-transcriptionally control gene expression in many pathogenic and physiological processes, we investigated the regulation of miRNAs by KLF2 in endothelial cells. KLF2 binds to the promoter and induces a significant upregulation of the miR-143/145 cluster. Interestingly, miR-143/145 has been shown to control smooth muscle cell (SMC) phenotypes; therefore, we investigated the possibility of transport of these miRNAs between endothelial cells and SMCs. Indeed, extracellular vesicles secreted by KLF2-transduced or shear-stress-stimulated HUVECs are enriched in miR-143/145 and control target gene expression in co-cultured SMCs. Extracellular vesicles derived from KLF2-expressing endothelial cells also reduced atherosclerotic lesion formation in the aorta of ApoE(-/-) mice. Combined, our results show that atheroprotective stimuli induce communication between endothelial cells and SMCs through an miRNA- and extracellular-vesicle-mediated mechanism and that this may comprise a promising strategy to combat atherosclerosis. (hide)
EV-METRIC
29% (68th 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
Protein markers
EV: None
non-EV: None
Proteomics
yes
TEM measurements
60-130(99median)
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
cryo EM
Image type
Close-up
EV120057 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Ekström K 2012 29%

Study summary

Full title
All authors
Ekström K, Valadi H, Sjöstrand M, Malmhäll C, Bossios A, Eldh M, Lötvall J
Journal
J Extracell Vesicles
Abstract
BACKGROUND: Exosomes are nanosized vesicles of endocytic origin that are released into the extracell (show more...)BACKGROUND: Exosomes are nanosized vesicles of endocytic origin that are released into the extracellular environment by many different cells. It has been shown that exosomes from various cellular origins contain a substantial amount of RNA (mainly mRNA and microRNA). More importantly, exosomes are capable of delivering their RNA content to target cells, which is a novel way of cell-to-cell communication. The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34(+) progenitor cells. METHODS: The mRNA and microRNA content of exosomes from a human mast cell line, HMC-1, was analysed by using microarray technology. Co-culture experiments followed by flow cytometry analysis and confocal microscopy as well as radioactive labeling experiments were performed to examine the uptake of these exosomes and the shuttle of the RNA to other mast cells and CD34(+) progenitor cells. RESULTS: In this study, we show that human mast cells release RNA-containing exosomes, with the capacity to shuttle RNA between cells. Interestingly, by using microRNA microarray analysis, 116 microRNAs could be identified in the exosomes and 134 microRNAs in the donor mast cells. Furthermore, DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. In addition, transfer experiments revealed that exosomes can shuttle RNA between human mast cells and to CD34(+) hematopoietic progenitor cells. CONCLUSION: These findings suggest that exosomal shuttle RNA (esRNA) can play a role in the communication between cells, including mast cells and CD34(+) progenitor cells, implying a role in cells maturation process. (hide)
EV-METRIC
29% (68th 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
130.7 (pelleting)
Protein markers
EV: CD63/ CD81/ CD9
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 ultra centrifugation
Differential UC: filtering 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
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
130.7
Filtration steps
0.22µm or 0.2µm
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
transmission EM
EV120108 1/2 Mesocricetus auratus Cell culture supernatant Density cushion Wik L 2012 25%

Study summary

Full title
All authors
Wik L, Klingeborn M, Willander H, Linne T
Journal
Prion
Abstract
The cellular prion protein (PrP (C) ) is attached to the cell membrane via its glycosylphosphatidyli (show more...)The cellular prion protein (PrP (C) ) is attached to the cell membrane via its glycosylphosphatidylinositol (GPI)-anchor and is constitutively shed into the extracellular space. Here, three different mechanisms are presented that concurrently shed PrP (C) from the cell. The fast ?-cleavage released a N-terminal fragment (N1) into the medium and the extreme C-terminal cleavage shed soluble full-length (FL-S) PrP and C-terminally cleaved (C1-S) fragments outside the cell. Also, a slow exosomal release of full-length (FL) and C1-fragment (C1) was demonstrated. The three separate mechanisms acting simultaneously, but with different kinetics, have to be taken into consideration when elucidating functional roles of PrP (C) and also when processing of PrP (C) is considered as a target for intervention in prion diseases. Further, in this study it was shown that metalloprotease inhibitors affected the extreme C-terminal cleavage and shedding of PrP (C) . The metalloprotease inhibitors did not influence the ?-cleavage or the exosomal release. Taken together, these results are important for understanding the different mechanisms acting in parallel in the shedding and cleavage of PrP (C) . (hide)
EV-METRIC
25% (64th 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
Density cushion
Protein markers
EV: TSG101/ Actin
non-EV: None
Proteomics
no
Show all info
Study aim
Other/The shedding and release mechanism of prion proteins
Sample
Species
Mesocricetus auratus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101/ Actin
ELISA
Detected EV-associated proteins
Actin
Characterization: Particle analysis
EM
EM-type
immune EM/ scanning EM
Image type
Close-up, Wide-field
EV120024 1/3 Homo sapiens Cell culture supernatant 0.1 µm filter
Immunoaffinity
Tauro BJ 2012 25%

Study summary

Full title
All authors
Tauro BJ, Greening DW, Mathias RA, Ji H, Mathivanan S, Scott AM, Simpson RJ
Journal
Methods
Abstract
Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and p (show more...)Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs. Using the colorectal cancer cell line LIM1863 as a cell model, in this study we performed a comprehensive evaluation of current methods used for exosome isolation including ultracentrifugation (UC-Exos), OptiPrep™ density-based separation (DG-Exos), and immunoaffinity capture using anti-EpCAM coated magnetic beads (IAC-Exos). Notably, all isolations contained 40-100nm vesicles, and were positive for exosome markers (Alix, TSG101, HSP70) based on electron microscopy and Western blotting. We employed a proteomic approach to profile the protein composition of exosomes, and label-free spectral counting to evaluate the effectiveness of each method. Based on the number of MS/MS spectra identified for exosome markers and proteins associated with their biogenesis, trafficking, and release, we found IAC-Exos to be the most effective method to isolate exosomes. For example, Alix, TSG101, CD9 and CD81 were significantly higher (at least 2-fold) in IAC-Exos, compared to UG-Exos and DG-Exos. Application of immunoaffinity capture has enabled the identification of proteins including the ESCRT-III component VPS32C/CHMP4C, and the SNARE synaptobrevin 2 (VAMP2) in exosomes for the first time. Additionally, several cancer-related proteins were identified in IAC-Exos including various ephrins (EFNB1, EFNB2) and Eph receptors (EPHA2-8, EPHB1-4), and components involved in Wnt (CTNNB1, TNIK) and Ras (CRK, GRB2) signalling. (hide)
EV-METRIC
25% (64th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.1 µm filter + Immunoaffinity
Protein markers
EV: Alix/ HSP70/ TSG101
non-EV: None
Proteomics
yes
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Filtration steps
0.2µm > x > 0.1µm
Immunoaffinity capture
Selected surface protein(s)
EpCAM
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ HSP70/ TSG101
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120046 2/2 Homo sapiens Cell culture supernatant Commercial Surgucheva I 2012 25%

Study summary

Full title
All authors
Surgucheva I, Sharov VS, Surguchov A
Journal
Biochemistry
Abstract
Protein misfolding and aggregation is a ubiquitous phenomenon associated with a wide range of diseas (show more...)Protein misfolding and aggregation is a ubiquitous phenomenon associated with a wide range of diseases. The synuclein family comprises three small naturally unfolded proteins implicated in neurodegenerative diseases and some forms of cancer. ?-Synuclein is a soluble protein that forms toxic inclusions associated with Parkinson's disease and several other synucleinopathies. However, the triggers inducing its conversion into noxious species are elusive. Here we show that another member of the family, ?-synuclein, can be easily oxidized and form annular oligomers that accumulate in cells in the form of deposits. Importantly, oxidized ?-synuclein can initiate ?-synuclein aggregation. Two amino acid residues in ?-synuclein, methionine and tyrosine located in neighboring positions (Met(38) and Tyr(39)), are most easily oxidized. Their oxidation plays a key role in the ability of ?-synuclein to aggregate and seed the aggregation of ?-synuclein. ?-Synuclein secreted from neuronal cells into conditioned medium in the form of exosomes can be transmitted to glial cells and cause the aggregation of intracellular proteins. Our data suggest that post-translationally modified ?-synuclein possesses prion-like properties and may induce a cascade of events leading to synucleinopathies. (hide)
EV-METRIC
25% (64th 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
Commercial
Protein markers
EV: CD63/ Alpha-synuclein
non-EV: None
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 ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Alpha-synuclein
ELISA
Detected EV-associated proteins
Alpha-synuclein
Characterization: Particle analysis
EV120045 1/1 Homo sapiens Urine UF Sun AL 2012 25%

Study summary

Full title
All authors
Sun AL, Deng JT, Guan GJ, Chen SH, Liu YT, Cheng J, Li ZW, Zhuang XH, Sun FD, Deng HP
Journal
Diab Vasc Dis Res
Abstract
The present study was designed to identify the changes in microvesicle-dipeptidyl peptidase-IV (DPP (show more...)The present study was designed to identify the changes in microvesicle-dipeptidyl peptidase-IV (DPP IV) levels in human urine and serum, and to determine whether there were correlations with the severity of diabetic kidney disease (DKD). A total of 127 patients with type 2 diabetes mellitus (T2DM) were divided into three groups according to the urinary albumin/ creatinine ratio (UACR): microalbuminuria group (n = 50); macroalbuminuria group (n = 34) and normoalbuminuria group (n = 43), and 34 age- and sex-matched non-diabetic healthy subjects were selected as controls. Microvesicle-bound DPP IV and free urinary DPP IV were separated by a filtra-centrifugation method. The total microvesicles were captured by a specific monoclonal antibody, AD-1. DPP IV activity was determined by measuring the cleavage of chromogenic free 4-nitroaniline from Gly-Pro-p-nitroanilide at 405 nm with an ELISA plate reader. DPP IV protein levels were determined by ELISA and Western blot. Our results showed that the microvesicle-bound type was the major form of DPP IV in urine; the urinary microvesicle-DPP IV excretion of each T2DM group was significantly higher compared with controls. The urinary microvesicle-DPP IV level was positively correlated with UACR in patients with T2DM. These findings suggest that the urinary level of microvesicle-bound DPP IV is associated with the severity of DKD. (hide)
EV-METRIC
25% (59th 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
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
UF
Protein markers
EV: "AD-1/ DPP IV"
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 10,000 g and 50,000 g
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
"AD-1/ DPP IV"
ELISA
Detected EV-associated proteins
"AD-1/ DPP IV"
Characterization: Particle analysis
EV120021 2/2 Homo sapiens Cell culture supernatant Commercial King HW 2012 25%

Study summary

Full title
All authors
King HW, Michael MZ, Gleadle JM
Journal
BMC Cancer
Abstract
BACKGROUND: Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signall (show more...)BACKGROUND: Exosomes are nanovesicles secreted by tumour cells which have roles in paracrine signalling during tumour progression, including tumour-stromal interactions, activation of proliferative pathways and bestowing immunosuppression. Hypoxia is an important feature of solid tumours which promotes tumour progression, angiogenesis and metastasis, potentially through exosome-mediated signalling. METHODS: Breast cancer cell lines were cultured under either moderate (1% O2) or severe (0.1% O2) hypoxia. Exosomes were isolated from conditioned media and quantitated by nanoparticle tracking analysis (NTA) and immunoblotting for the exosomal protein CD63 in order to assess the impact of hypoxia on exosome release. Hypoxic exosome fractions were assayed for miR-210 by real-time reverse transcription polymerase chain reaction and normalised to exogenous and endogenous control genes. Statistical significance was determined using the Student T test with a P value of < 0.05 considered significant. RESULTS: Exposure of three different breast cancer cell lines to moderate (1% O2) and severe (0.1% O2) hypoxia resulted in significant increases in the number of exosomes present in the conditioned media as determined by NTA and CD63 immunoblotting. Activation of hypoxic signalling by dimethyloxalylglycine, a hypoxia-inducible factor (HIF) hydroxylase inhibitor, resulted in significant increase in exosome release. Transfection of cells with HIF-1? siRNA prior to hypoxic exposure prevented the enhancement of exosome release by hypoxia. The hypoxically regulated miR-210 was identified to be present at elevated levels in hypoxic exosome fractions. CONCLUSIONS: These data provide evidence that hypoxia promotes the release of exosomes by breast cancer cells, and that this hypoxic response may be mediated by HIF-1?. Given an emerging role for tumour cell-derived exosomes in tumour progression, this has significant implications for understanding the hypoxic tumour phenotype, whereby hypoxic cancer cells may release more exosomes into their microenvironment to promote their own survival and invasion. (hide)
EV-METRIC
25% (64th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial
Protein markers
EV: CD63/ CD9
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 ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD9
Characterization: Particle analysis
NTA
EM
EM-type
immune EM
Image type
Close-up
EV120067 1/2 Homo sapiens Serum Commercial Khan S 2012 25%

Study summary

Full title
All authors
Khan S, Jutzy JM, Valenzuela MM, Turay D, Aspe JR, Ashok A, Mirshahidi S, Mercola D, Lilly MB, Wall NR
Journal
PLoS One
Abstract
BACKGROUND: Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa ce (show more...)BACKGROUND: Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment. METHODS: Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively. RESULTS: Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six) or high (nine) Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls. CONCLUSIONS: These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection. (hide)
EV-METRIC
25% (80th 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
Commercial
Protein markers
EV: LAMP1/ AChE
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
Isolation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
LAMP1/ AChE
ELISA
Detected EV-associated proteins
LAMP1/ AChE
Characterization: Particle analysis
EV120061 1/1 Homo sapiens Serum SEC
UF
Gercel-Taylor C 2012 25%

Study summary

Full title
All authors
Gercel-Taylor C, Atay S, Tullis RH, Kesimer M, Taylor DD
Journal
Anal Biochem
Abstract
Cell-derived vesicles are recognized as essential components of intercellular communication, and man (show more...)Cell-derived vesicles are recognized as essential components of intercellular communication, and many disease processes are associated with their aberrant composition and release. Circulating tumor-derived vesicles have major potential as biomarkers; however, the diagnostic use of exosomes is limited by the technology available for their objective characterization and measurement. In this study, we compare nanoparticle tracking analysis (NTA) with submicron particle analysis (SPA), dynamic light scattering (DLS), and electron microscopy (EM) to objectively define size distribution, number, and phenotype of circulating cell-derived vesicles from ovarian cancer patients. Using the NanoSight LM10 instrument, cell-derived vesicles were visualized by laser light scattering and analyzing Brownian motion of these vesicles captured by video. The NTA software calculates the size and total concentration of the vesicles in solution. Using vesicles isolated from ovarian cancer patients, we demonstrate that NTA can measure the size distributions of cell-derived vesicles comparable to other analysis instrumentation. Size determinations by NTA, SPA, and DLS were more objective and complete than that obtained with the commonly used EM approach. NTA can also define the total vesicle concentration. Furthermore, the use of fluorescent-labeled antibodies against specific markers with NTA allows the determination of the phenotype of the cell-derived vesicles. (hide)
EV-METRIC
25% (80th 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
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
SEC + UF
Protein markers
EV: CD63
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Serum
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63
Characterization: Particle analysis
DLS
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV120112 3/4 Equus caballus Other Commercial da Silveira JC 2012 25%

Study summary

Full title
All authors
da Silveira JC, Veeramachaneni DN, Winger QA, Carnevale EM, Bouma GJ
Journal
Biol Reprod
Abstract
Proper cell communication within the ovarian follicle is critical for the growth and maturation of a (show more...)Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid. (hide)
EV-METRIC
25% (60th 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
Other
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
Commercial
Protein markers
EV: HSP70
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Equus caballus
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
HSP70
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120097 1/2 Homo sapiens Cell culture supernatant dUC
UF
Xiao D 2012 22%

Study summary

Full title
All authors
Xiao D, Ohlendorf J, Chen Y, Taylor DD, Rai SN, Waigel S, Zacharias W, Hao H, McMasters KM
Journal
PLoS One
Abstract
BACKGROUND: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, (show more...)BACKGROUND: Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma. (hide)
EV-METRIC
22% (56th 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 + UF
Protein markers
EV: CD81/ Syntenin/ HSC70/ Annexin2
non-EV: Cell organelle protein
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ Syntenin/ HSC70/ Annexin2
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
HSC70/ Annexin2
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120089 1/1 Borrelia burgdorferi Other 0.2 µm filter
dUC
Iodixanol-DG (valid.)
Toledo A 2012 22%

Study summary

Full title
All authors
Toledo A, Coleman JL, Kuhlow CJ, Crowley JT, Benach JL
Journal
Infect Immun
Abstract
The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are di (show more...)The agent of Lyme disease, Borrelia burgdorferi, has a number of outer membrane proteins that are differentially regulated during its life cycle. In addition to their physiological functions in the organism, these proteins also likely serve different functions in invasiveness and immune evasion. In borreliae, as well as in other bacteria, a number of membrane proteins have been implicated in binding plasminogen. The activation and transformation of plasminogen into its proteolytically active form, plasmin, enhances the ability of the bacteria to disseminate in the host. Outer membrane vesicles of B. burgdorferi contain enolase, a glycolytic-cycle enzyme that catalyzes 2-phosphoglycerate to form phosphoenolpyruvate, which is also a known plasminogen receptor in Gram-positive bacteria. The enolase was cloned, expressed, purified, and used to generate rabbit antienolase serum. The enolase binds plasminogen in a lysine-dependent manner but not through ionic interactions. Although it is present in the outer membrane, microscopy and proteinase K treatment showed that enolase does not appear to be exposed on the surface. However, enolase in the outer membrane vesicles is accessible to proteolytic degradation by proteinase K. Samples from experimentally and tick-infected mice and rabbits as well as from Lyme disease patients exhibit recognition of enolase in serologic assays. Thus, this immunogenic plasminogen receptor released in outer membrane vesicles could be responsible for external proteolysis in the pericellular environment and have roles in nutrition and in enhancing dissemination. (hide)
EV-METRIC
22% (55th 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
Other
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 + dUC + Iodixanol-DG (valid.)
Protein markers
EV: "OspA/ Enolase"
non-EV: DnaK
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Borrelia burgdorferi
Sample Type
Other
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
15
Highest density fraction
40
Orientation
Bottom-up
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
"OspA/ Enolase"
Detected contaminants
DnaK
ELISA
Detected EV-associated proteins
"OspA/ Enolase"
Characterization: Particle analysis
EV120046 1/2 Homo sapiens Cell culture supernatant dUC Surgucheva I 2012 22%

Study summary

Full title
All authors
Surgucheva I, Sharov VS, Surguchov A
Journal
Biochemistry
Abstract
Protein misfolding and aggregation is a ubiquitous phenomenon associated with a wide range of diseas (show more...)Protein misfolding and aggregation is a ubiquitous phenomenon associated with a wide range of diseases. The synuclein family comprises three small naturally unfolded proteins implicated in neurodegenerative diseases and some forms of cancer. ?-Synuclein is a soluble protein that forms toxic inclusions associated with Parkinson's disease and several other synucleinopathies. However, the triggers inducing its conversion into noxious species are elusive. Here we show that another member of the family, ?-synuclein, can be easily oxidized and form annular oligomers that accumulate in cells in the form of deposits. Importantly, oxidized ?-synuclein can initiate ?-synuclein aggregation. Two amino acid residues in ?-synuclein, methionine and tyrosine located in neighboring positions (Met(38) and Tyr(39)), are most easily oxidized. Their oxidation plays a key role in the ability of ?-synuclein to aggregate and seed the aggregation of ?-synuclein. ?-Synuclein secreted from neuronal cells into conditioned medium in the form of exosomes can be transmitted to glial cells and cause the aggregation of intracellular proteins. Our data suggest that post-translationally modified ?-synuclein possesses prion-like properties and may induce a cascade of events leading to synucleinopathies. (hide)
EV-METRIC
22% (56th 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
Protein markers
EV: CD63/ Alpha-synuclein
non-EV: None
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 ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Alpha-synuclein
ELISA
Detected EV-associated proteins
Alpha-synuclein
Characterization: Particle analysis
EV120043 1/1 Homo sapiens Cell culture supernatant dUC
Immunoaffinity (valid.)
Soo CY 2012 22%

Study summary

Full title
All authors
Soo CY, Song Y, Zheng Y, Campbell EC, Riches AC, Gunn-Moore F, Powis SJ
Journal
Immunology
Abstract
Nanoparticle tracking analysis permits the determination of both the size distribution and relative (show more...)Nanoparticle tracking analysis permits the determination of both the size distribution and relative concentration of microvesicles, including exosomes, in the supernatants of cultured cells and biological fluids. We have studied the release of microvesicles from the human lymphoblastoid T-cell lines Jurkat and CEM. Unstimulated, both cell lines release microvesicles in the size range 70-90 nm, which can be depleted from the supernatant by ultracentrifugation at 100 000 g, and by anti-CD45 magnetic beads, and which by immunoblotting also contain the exosome-associated proteins Alix and Tsg101. Incubation with known potentiators of exosome release, the ionophores monensin and A23187, resulted in a significant increase in microvesicle release that was both time and concentration dependent. Mass spectrometric analysis of proteins isolated from ultracentrifuged supernatants of A23187-treated cells revealed the presence of exosome-associated proteins including heat-shock protein 90, tubulin, elongation factor ?1, actin and glyceraldehyde 3-phosphate dehydrogenase. Additionally, treatment of peripheral blood monocyte-derived dendritic cells with bacterial lipopolysaccharide displayed an increase in secreted microvesicles. Consequently, nanoparticle tracking analysis can be effectively applied to monitor microvesicle release from cells of the immune system. (hide)
EV-METRIC
22% (56th 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 / 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.)
Protein markers
EV: Alix/ TSG101/ CD45/ MHC1
non-EV: Cell organelle protein
Proteomics
yes
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Immunoaffinity capture
Selected surface protein(s)
CD45
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ CD45/ MHC1
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
CD45/ MHC1
Characterization: Particle analysis
NTA
EV120086 2/2 Homo sapiens Blood plasma 0.2 µm filter
dUC
Silva J 2012 22%

Study summary

Full title
All authors
Silva J, Garcia V, Rodriguez M, Compte M, Cisneros E, Veguillas P, Garcia JM, Dominguez G, Campos-Martin Y, Cuevas J, Peña C, Herrera M, Diaz R, Mohammed N, Bonilla F
Journal
Genes Chromosomes Cancer
Abstract
A significant proportion of extracellular nucleic acids in plasma circulate highly protected in tumo (show more...)A significant proportion of extracellular nucleic acids in plasma circulate highly protected in tumor-specific exosomes, but it is unclear how the release of exosomes is modulated in carcinogenesis. We quantified by cytometry exosomes in plasma of 91 colorectal cancer patients to evaluate their potential as a tumor indicator and their repercussions on diagnosis and prognosis. We examined the involvement of TSAP6, a TP53-regulated gene involved in the regulation of vesicular secretion, in levels of circulating exosomes in plasma of colorectal patients and in HCT116 TP53-(wild-type and null) human colorectal cancer cell lines. The fraction of exosomes in cancer patients was statistically higher than in healthy controls (mean rank ¼ 53.93 vs. 24.35). High levels of exosomes in plasma of patients correlated with high levels of carcino-embryonic antigen (P ¼ 0.029) and with poorly differentiated tumors (P ¼ 0.039) and tended to have shorter overall survival than patients with low levels (P ¼ 0.056). Release of exosomes did not correlate with TSAP6 expression; and regulation of TSAP6 by TP53 was not shown either in tumor samples or in HCT116 cell lines. Although it was not suggested that the TP53/TSAP6 pathway regulates the release of exosomes into the plasma of colorectal cancer patients, the level of circulating exosomes may be used as a tumor indicator, because it correlates with poor prognosis parameters and shorter survival. (hide)
EV-METRIC
22% (64th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Protein markers
EV: CD63/ Flotilin1/ EpCAM
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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/ Flotilin1/ EpCAM
ELISA
Detected EV-associated proteins
EpCAM
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EV120040 1/1 Rattus norvegicus/rattus Cell culture supernatant Density cushion
dUC
Rana S 2012 22%

Study summary

Full title
All authors
Rana S, Yue S, Stadel D, Zöller M
Journal
Int J Biochem Cell Biol
Abstract
Exosomes are discussed as potent therapeutics due to efficient transfer of proteins, mRNA and miRNA (show more...)Exosomes are discussed as potent therapeutics due to efficient transfer of proteins, mRNA and miRNA in selective targets. However, therapeutic exosome application requires knowledge on target structures to avoid undue delivery. Previous work suggesting exosomal tetraspanin-integrin complexes to be involved in target cell binding, we aimed to control this hypothesis and to define target cell ligands. Exosomes are rich in tetraspanins that associate besides other molecules with integrins. Co-immunoprecipitation of exosome lysates from rat tumor lines that differ only with respect to Tspan8 and beta4 revealed promiscuity of tetraspanin-integrin associations, but also few preferential interactions like that of Tspan8 with alpha4 and beta4 integrin chains. These minor differences in exosomal tetraspanin-complexes strongly influence target cell selection in vitro and in vivo, efficient exosome-uptake being seen in hematopoietic cells and solid organs. Exosomes expressing the Tspan8-alpha4 complex are most readily taken up by endothelial and pancreas cells, CD54 serving as a major ligand. Selectivity of uptake was confirmed with exosomes from an alpha4 cDNA transfected Tspan8(+) lymph node stroma line. Distinct from exosomes from the parental line, the latter preferentially targeted endothelial cells and in vivo the pancreas. Importantly, pulldown experiments provided strong evidence that exosome-uptake occurs in internalization-prone membrane domains. This is the first report on the exosomal tetraspanin web contributing to target cell selection such that predictions can be made on potential targets, which will facilitate tailoring exosomes for drug delivery. (hide)
EV-METRIC
22% (56th 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
Density cushion + dUC
Protein markers
EV: CD9/ HSP70/ Clusterin/ GDF15/ Neuropilin/ Thrombospondin/ Rab7/ Tspan/ Annexin2/ Tubulin/ Clathrin
non-EV: Cell organelle protein
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering 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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ HSP70/ Clusterin/ GDF15/ Neuropilin/ Thrombospondin/ Rab7/ Tspan/ Annexin2/ Tubulin/ Clathrin
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Clusterin/ GDF15/ Neuropilin/ Thrombospondin/ Rab7/ Tspan/ Annexin2/ Tubulin/ Clathrin
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EV120083 1/1 Homo sapiens Urine Density cushion
dUC
Raj DA 2012 22%

Study summary

Full title
All authors
Raj DA, Fiume I, Capasso G, Pocsfalvi G
Journal
Kidney Int
Abstract
Urinary exosomes have received considerable attention as a potential biomarker source for the diagno (show more...)Urinary exosomes have received considerable attention as a potential biomarker source for the diagnosis of renal diseases. Notwithstanding, their use in protein biomarker research is hampered by the lack of efficient methods for vesicle isolation, lysis, and protein quantification. Here we report an improved ultracentrifugation-based method that facilitates the solubilization and removal of major impurities associated with urinary exosomes. A double-cushion sucrose/D(2)O centrifugation step was used after a two-step differential centrifugation to separate exosomes from the heavier vesicles. After the removal of uromodulin, 378 and 79 unique proteins were identified, respectively, in low- and high-density fractions. Comparison of our data with two previously published data sets helped to define proteins commonly found in urinary exosomes. Lysis, protein extraction, and in-solution digestion of exosomes were then optimized for MudPIT application. More than a hundred exosomal proteins were quantified by four-plex iTRAQ analysis of single and pooled samples from two different age groups. For healthy men, six proteins (TSN1, PODXL, IDHC, PPAP, ACBP, and ANXA5) showed significant expression differences between exosome pools of those aged 25-50 and 50-70 years old. Thus, exosomes isolated by our method provide the basis for the development of robust quantitative methods for protein biomarker research. (hide)
EV-METRIC
22% (53rd 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
Density cushion + dUC
Adj. k-factor
78.45 (pelleting)
Protein markers
EV: Alix/ CD9/ TSG101/ AQP2/ Beta-actin
non-EV: None
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
78.45
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD9/ TSG101/ AQP2/ Beta-actin
ELISA
Detected EV-associated proteins
AQP2/ Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120009 1/2 Homo sapiens Cell culture supernatant dUC Putz U 2012 22%

Study summary

Full title
All authors
Putz U, Howitt J, Doan A, Goh CP, Low LH, Silke J, Tan SS
Journal
Sci Signal
Abstract
Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipi (show more...)Exosomes are microvesicles of endosomal origin that are secreted, and their contents (proteins, lipids, DNA, or microRNAs) can alter the physiological states of recipient cells. We demonstrated that phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein normally localized in the cytoplasm and nucleus, was secreted in exosomes. Secreted PTEN was internalized by recipient cells with resultant functional activity, which resulted in reduced phosphorylation of the serine and threonine kinase Akt and reduced cellular proliferation. PTEN secretion in exosomes required Ndfip1, an adaptor protein for members of the Nedd4 family of E3 ubiquitin ligases. Without Ndfip1, neither Nedd4-1 nor Nedd4-2 promoted the recruitment of PTEN into exosomes. In addition, lysine 13 within PTEN, which is required for its ubiquitination by Nedd4-1, was required for exosomal transport of PTEN. These results implicate Ndfip1 as a molecular regulator of the exosomal export of PTEN, with consequences for non-cell-autonomous PTEN activity. Thus, we suggest that the ability of PTEN to exert phosphatase activity beyond the cell in which it is produced has implications for PTEN function during development, health, and disease. (hide)
EV-METRIC
22% (56th 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