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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
EV120007 1/2 Homo sapiens Cell culture supernatant dUC
Sucrose-DG
Lugini L 2012 78%

Study summary

Full title
All authors
Lugini L, Cecchetti S, Huber V, Luciani F, Macchia G, Spadaro F, Paris L, Abalsamo L, Colone M, Molinari A, Podo F, Rivoltini L, Ramoni C, Fais S
Journal
J Immunol
Abstract
Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture s (show more...)Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by normal cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors. (hide)
EV-METRIC
78% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Adj. k-factor
41.45 (pelleting)
Protein markers
EV: CD63/ Rab5b/ MHC1/ CD56/ NKG2D
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.1-1.19
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential 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
TLA100.2
Pelleting: adjusted k-factor
41.45
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Rotor type
SW41
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Rab5b/ MHC1/ CD56/ NKG2D
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Rab5b/ MHC1/ CD56/ NKG2D
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up, Wide-field
EV120001 1/1 Oryctolagus cuniculus Cell culture supernatant 0.2 µm filter
Density cushion (valid.)
dUC
Iodixanol-DG (valid.)
Coleman BM 2012 78%

Study summary

Full title
All authors
Coleman BM, Hanssen E, Lawson VA, Hill AF
Journal
FASEB J
Abstract
Exosomes are small membrane-bound vesicles released from cells and found in vivo in most biological (show more...)Exosomes are small membrane-bound vesicles released from cells and found in vivo in most biological fluids. Functions reported for exosomes include cell-cell communication, roles in modulating immune responses, and roles in the transfer of pathogens such as prions. Here we investigated the molecular characteristics of the structure of exosomes that harbor prion infectivity to determine the native structure of exosomes and whether infected exosomes have a distinct structure. Cryo-electron tomography revealed the previously unidentified ultrastructural detail of exosomes with high resolution. Exosomes were found to be naturally spherical in shape and to have a diverse population that varies in size and internal structure, such as differences in the number of membrane structures. Exosomes isolated from prion-infected cells contained a significantly different population of exosomes with distinct structural features compared to control vesicles from mock-infected cells. Exosomes are highly structured vesicles that can modify their structure on altering their protein cargo. This finding provides further insight into the role that the exosomal protein cargo plays on influencing the structure of the vesicles as well as highlighting the diversity of exosomes and their relationship to biological processes. (hide)
EV-METRIC
78% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Density cushion (valid.) + dUC + Iodixanol-DG (valid.)
Protein markers
EV: CD63/ Flotilin1/ TSG101
non-EV: Cell organelle protein/ Bcl2
Proteomics
no
TEM measurements
112+-12.7
Show all info
Study aim
Other/Ultrastructure
Sample
Species
Oryctolagus cuniculus
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
Density gradient
Only used for validation of main results
1
Lowest density fraction
10
Highest density fraction
30
Orientation
Top-down
Speed (g)
10000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ TSG101
Detected contaminants
Cell organelle protein/ Bcl2
Characterization: Particle analysis
EM
EM-type
transmission EM/ cryo EM
Image type
Close-up, Wide-field
EV120003 1/2 Trypanosoma cruzi Other 0.45 µm filter
dUC
Sucrose-DG (valid.)
Bayer-Santos E 2012 78%

Study summary

Full title
All authors
Bayer-Santos E, Aguilar-Bonavides C, Rodrigues SP, Cordero EM, Marques AF, Varela-Ramirez A, Choi H, Yoshida N, da Silveira JF, Almeida IC
Journal
J Proteome Res
Abstract
Microorganisms use specialized systems to export virulence factors into host cells. Secretion of eff (show more...)Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells. (hide)
EV-METRIC
78% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Other
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + dUC + Sucrose-DG (valid.)
Adj. k-factor
240.8 (pelleting)
Protein markers
EV: GP82/ GP35/50/ FCaBP
non-EV: None
Proteomics
yes
EV density (g/ml)
1.08-1.14
Show all info
Study aim
Omics
Sample
Species
Trypanosoma cruzi
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)
120
Pelleting: rotor type
TH641
Pelleting: adjusted k-factor
240.8
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
200000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
GP82/ GP35/50/ FCaBP
ELISA
Detected EV-associated proteins
GP82/ GP35/50/ FCaBP
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120003 2/2 Trypanosoma cruzi Other 0.45 µm filter
dUC
Sucrose-DG (valid.)
Bayer-Santos E 2012 78%

Study summary

Full title
All authors
Bayer-Santos E, Aguilar-Bonavides C, Rodrigues SP, Cordero EM, Marques AF, Varela-Ramirez A, Choi H, Yoshida N, da Silveira JF, Almeida IC
Journal
J Proteome Res
Abstract
Microorganisms use specialized systems to export virulence factors into host cells. Secretion of eff (show more...)Microorganisms use specialized systems to export virulence factors into host cells. Secretion of effector proteins into the extracellular environment has been described in Trypanosoma cruzi; however, a comprehensive proteomic analysis of the secretome and the secretion mechanisms involved remain elusive. Here, we present evidence that T. cruzi releases proteins associated with vesicles that are formed by at least two different mechanisms. Transmission electron microscopy showed larger vesicles budding from the plasma membrane of noninfective epimastigotes and infective metacyclic trypomastigotes, as well as smaller vesicles within the flagellar pocket of both forms. Parasite conditioned culture supernatant was fractionated and characterized by morphological, immunochemical, and proteomic analyses. Three fractions were obtained by differential ultracentrifugation: the first enriched in larger vesicles resembling ectosomes, the second enriched in smaller vesicles resembling exosomes, and a third fraction enriched in soluble proteins not associated with extracellular vesicles. Label-free quantitative proteomic analysis revealed a rich collection of proteins involved in metabolism, signaling, nucleic acid binding, and parasite survival and virulence. These findings support the notion that T. cruzi uses different secretion pathways to excrete/secrete proteins. Moreover, our results suggest that metacyclic forms may use extracellular vesicles to deliver cargo into host cells. (hide)
EV-METRIC
78% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Other
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + dUC + Sucrose-DG (valid.)
Adj. k-factor
240.8 (pelleting)
Protein markers
EV: GP82/ GP35/50/ FCaBP
non-EV: None
Proteomics
yes
EV density (g/ml)
1.14-1.2
Show all info
Study aim
Omics
Sample
Species
Trypanosoma cruzi
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)
960
Pelleting: rotor type
TH641
Pelleting: adjusted k-factor
240.8
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
200000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
GP82/ GP35/50/ FCaBP
ELISA
Detected EV-associated proteins
GP82/ GP35/50/ FCaBP
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120009 2/2 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Putz U 2012 67%

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
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: Alix/ Flotilin1/ HSP90/ TSG101/ Beta-actin
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.08-1.14
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Flotilin1/ HSP90/ TSG101/ Beta-actin
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120007 2/2 Homo sapiens Blood plasma 0.2 µm filter
dUC
Sucrose-DG
Lugini L 2012 67%

Study summary

Full title
All authors
Lugini L, Cecchetti S, Huber V, Luciani F, Macchia G, Spadaro F, Paris L, Abalsamo L, Colone M, Molinari A, Podo F, Rivoltini L, Ramoni C, Fais S
Journal
J Immunol
Abstract
Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture s (show more...)Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by normal cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG
Adj. k-factor
37.69 (pelleting)
Protein markers
EV: Rab5b/ Perforin/ CD56/ NKG2D
non-EV: "NKp46/ NKp44/ CD3/ CD4/ CD8"
Proteomics
no
EV density (g/ml)
1.19-1.23
Show all info
Study aim
Function
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)
120
Pelleting: rotor type
TLA100.2
Pelleting: adjusted k-factor
37.69
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Rotor type
SW41
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Rab5b/ Perforin/ CD56/ NKG2D
Detected contaminants
"NKp46/ NKp44/ CD3/ CD4/ CD8"
ELISA
Detected EV-associated proteins
Rab5b/ Perforin/ CD56/ NKG2D
Characterization: Particle analysis
EV120004 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
de Jong OG 2012 67%

Study summary

Full title
All authors
de Jong OG, Verhaar MC, Chen Y, Vader P, Gremmels H, Posthuma G, Schiffelers RM, Gucek M, van Balkom BW
Journal
J Extracell Vesicles
Abstract
BACKGROUND: The healthy vascular endothelium, which forms the barrier between blood and the surround (show more...)BACKGROUND: The healthy vascular endothelium, which forms the barrier between blood and the surrounding tissues, is known to efficiently respond to stress signals like hypoxia and inflammation by adaptation of cellular physiology and the secretion of (soluble) growth factors and cytokines. Exosomes are potent mediators of intercellular communication. Their content consists of RNA and proteins from the cell of origin, and thus depends on the condition of these cells at the time of exosome biogenesis. It has been suggested that exosomes protect their target cells from cellular stress through the transfer of RNA and proteins. We hypothesized that endothelium-derived exosomes are involved in the endothelial response to cellular stress, and that exosome RNA and protein content reflect the effects of cellular stress induced by hypoxia, inflammation or hyperglycemia. METHODS: We exposed cultured endothelial cells to different types of cellular stress (hypoxia, TNF-?-induced activation, high glucose and mannose concentrations) and compared mRNA and protein content of exosomes produced by these cells by microarray analysis and a quantitative proteomics approach. RESULTS: We identified 1,354 proteins and 1,992 mRNAs in endothelial cell-derived exosomes. Several proteins and mRNAs showed altered abundances after exposure of their producing cells to cellular stress, which were confirmed by immunoblot or qPCR analysis. CONCLUSION: Our data show that hypoxia and endothelial activation are reflected in RNA and protein exosome composition, and that exposure to high sugar concentrations alters exosome protein composition only to a minor extend, and does not affect exosome RNA composition. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Adj. k-factor
256 (pelleting)
Protein markers
EV: CD63/ Flotilin1/ HSP70/ Beta-actin/ ICAM1
non-EV: None
Proteomics
yes
EV density (g/ml)
1.09-1.12
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
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
SW32;SW60
Pelleting: adjusted k-factor
256.0
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ HSP70/ Beta-actin/ ICAM1
ELISA
Detected EV-associated proteins
Beta-actin/ ICAM1
Characterization: Particle analysis
NTA
EM
EM-type
immune EM
Image type
Wide-field
EV120002 1/1 Homo sapiens Cell culture supernatant dUC
Iodixanol-DG (valid.)
Baietti MF 2012 67%

Study summary

Full title
All authors
Baietti MF, Zhang Z, Mortier E, Melchior A, Degeest G, Geeraerts A, Ivarsson Y, Depoortere F, Coomans C, Vermeiren E, Zimmermann P, David G
Journal
Nat Cell Biol
Abstract
The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incomp (show more...)The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes. (hide)
EV-METRIC
67% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Iodixanol-DG (valid.)
Protein markers
EV: Alix/ CD63/ Flotilin1/ HSP70/ Syntenin/ TSG101/ Syndecan1
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.094-1.143
Show all info
Study aim
Biogenesis/Sorting
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)
180
Density gradient
Only used for validation of main results
1
Lowest density fraction
6
Highest density fraction
40
Orientation
Bottom-up
Speed (g)
140000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ Flotilin1/ HSP70/ Syntenin/ TSG101/ Syndecan1
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Syndecan1
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up
EV120008 2/2 Mus musculus Cell culture supernatant Sucrose-DG (valid.)
UF
Montecalvo A 2012 63%

Study summary

Full title
All authors
Montecalvo A, Larregina AT, Shufesky WJ, Stolz DB, Sullivan ML, Karlsson JM, Baty CJ, Gibson GA, Erdos G, Wang Z, Milosevic J, Tkacheva OA, Divito SJ, Jordan R, Lyons-Weiler J, Watkins SC, Morelli AE
Journal
Blood
Abstract
Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses (show more...)Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs. (hide)
EV-METRIC
63% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Sucrose-DG (valid.) + UF
Protein markers
EV: CD81/ CD9/ MHC1/ MHC2/ CD86/ CD54
non-EV: None
Proteomics
no
EV density (g/ml)
1.166-1.199
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
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Speed (g)
200000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ CD9/ MHC1/ MHC2/ CD86/ CD54
ELISA
Detected EV-associated proteins
CD81/ CD9/ MHC1/ MHC2/ CD86/ CD54
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV120025 2/3 Homo sapiens Other Density cushion
SEC
Sucrose-DG (valid.)
Aalberts M 2012 63%

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
63% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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/ PSCA
non-EV: None
Proteomics
yes
EV density (g/ml)
1.12-1.16;1.23-1.26
TEM measurements
56+-13 (high density);105+-25 (low density)
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
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ PSCA
ELISA
Detected EV-associated proteins
PSCA
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM/ immune EM
Image type
Wide-field
EV120037 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG
Pan Q 2012 57%

Study summary

Full title
All authors
Pan Q, Ramakrishnaiah V, Henry S, Fouraschen S, de Ruiter PE, Kwekkeboom J, Tilanus HW, Janssen HL, van der Laan LJ
Journal
Gut
Abstract
BACKGROUND/AIMS: RNA interference (RNAi), a sequence-specific gene silencing technology triggered by (show more...)BACKGROUND/AIMS: RNA interference (RNAi), a sequence-specific gene silencing technology triggered by small interfering RNA (siRNA), represents promising new avenues for treatment of various liver diseases including hepatitis C virus (HCV) infection. In plants and invertebrates, RNAi provides an important mechanism of cellular defence against viral pathogens and is dependent on the spread of siRNA to neighbouring cells. A study was undertaken to investigate whether vector-delivered RNAi can transfer between hepatic cells in vitro and in mice, and whether this exchange could extend the therapeutic effect of RNAi against HCV infection. METHODS: Transmission of RNAi was investigated in culture by assessing silencing of HCV replication and expression of viral entry receptor CD81 using a human hepatic cell line and primary B lymphocytes transduced with siRNA-expressing vectors. In vivo transmission between hepatic cells was investigated in NOD/SCID mice. Involvement of exosomes was demonstrated by purification, uptake and mass spectrometric analysis. RESULTS: Human and mouse liver cells, as well as primary human B cells, were found to have the ability to exchange small RNAs, including cellular endogenous microRNA and delivered siRNA targeting HCV or CD81. The transmission of RNAi was largely independent of cell contact and partially mediated by exosomes. Evidence of RNAi transmission in vivo was observed in NOD/SCID mice engrafted with human hepatoma cells producing CD81 siRNA, causing suppression of CD81 expression in mouse hepatocytes. CONCLUSION: Both human and mouse hepatic cells exchange small silencing RNAs, partially mediated by shuttling of exosomes. Transmission of siRNA potentially extends the therapeutic reach of RNAi-based therapies against HCV as well as other liver diseases. (hide)
EV-METRIC
57% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Adj. k-factor
396.5 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
yes
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
Between 50,000 g and 100,000 g
Pelleting: time(min)
110
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
396.5
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120023 1/3 Homo sapiens Cell culture supernatant 0.45 µm filter
dUC
Sucrose-DG
Palma J 2012 57%

Study summary

Full title
All authors
Palma J, Yaddanapudi SC, Pigati L, Havens MA, Jeong S, Weiner GA, Weimer KM, Stern B, Hastings ML, Duelli DM
Journal
Nucleic Acids Res
Abstract
MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previou (show more...)MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body. (hide)
EV-METRIC
57% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
Particles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + dUC + Sucrose-DG
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.13-1.19
TEM measurements
86
Show all info
Study aim
Biomarker
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 50,000 g and 100,000 g
Pelleting: time(min)
60
Density gradient
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120011 1/1 Homo sapiens Cell culture supernatant dUC Andersson-Willman B 2012 57%

Study summary

Full title
All authors
Andersson-Willman B, Gehrmann U, Cansu Z, Buerki-Thurnherr T, Krug HF, Gabrielsson S, Scheynius A
Journal
Toxicol Appl Pharmacol
Abstract
Metal oxide nanoparticles are widely used in the paint and coating industry as well as in cosmetics, (show more...)Metal oxide nanoparticles are widely used in the paint and coating industry as well as in cosmetics, but the knowledge of their possible interactions with the immune system is very limited. Our aims were to investigate if commercially available TiO(2) and ZnO nanoparticles may affect different human immune cells and their production of exosomes, nano-sized vesicles that have a role in cell to cell communication. We found that the TiO(2) or ZnO nanoparticles at concentrations from 1 to 100?g/mL did not affect the viability of primary human peripheral blood mononuclear cells (PBMC). In contrast, monocyte-derived dendritic cells (MDDC) reacted with a dose dependent increase in cell death and caspase activity to ZnO but not to TiO(2) nanoparticles. Non-toxic exposure, 10?g/mL, to TiO(2) and ZnO nanoparticles did not significantly alter the phenotype of MDDC. Interestingly, ZnO but not TiO(2) nanoparticles induced a down regulation of Fc?RIII (CD16) expression on NK-cells in the PBMC population, suggesting that subtoxic concentrations of ZnO nanoparticles might have an effect on Fc?R-mediated immune responses. The phenotype and size of exosomes produced by PBMC or MDDC exposed to the nanoparticles were similar to that of exosomes harvested from control cultures. TiO(2) or ZnO nanoparticles could not be detected within or associated to exosomes as analyzed with TEM. We conclude that TiO(2) and ZnO nanoparticles differently affect immune cells and that evaluations of nanoparticles should be performed even at subtoxic concentrations on different primary human immune cells when investigating potential effects on immune functions. (hide)
EV-METRIC
57% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
209.7 (pelleting) / 53.3 (washing)
Protein markers
EV: CD63/ CD81/ MHC2/ MHC1
non-EV: None
Proteomics
no
Show all info
Study aim
Other/Effect of metal oxide nanoparticles on released exosomes
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)
130
Pelleting: rotor type
45Ti
Pelleting: adjusted k-factor
209.7
Wash: Rotor Type
NVT90
Wash: adjusted k-factor
53.3
Western Blot
Detected EV-associated proteins
MHC2/ MHC1
ELISA
Detected EV-associated proteins
MHC2/ MHC1
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
NTA
Particle analysis: flow cytometry
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120047 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG
Wan C 2012 56%

Study summary

Full title
All authors
Wan C, Fu J, Wang Y, Miao S, Song W, Wang L
Journal
PLoS One
Abstract
We have previously reported that rhomboid domain containing 1 (RHBDD1), a mammalian rhomboid proteas (show more...)We have previously reported that rhomboid domain containing 1 (RHBDD1), a mammalian rhomboid protease highly expressed in the testis, can cleave the Bcl-2 protein Bik. In this study, we identified a multi-pass transmembrane protein, tumor suppressor activated pathway-6 (TSAP6) as a potential substrate of RHBDD1. RHBDD1 was found to induce the proteolysis of TSAP6 in a dose- and activity-dependent manner. The cleavage of TSAP6 was not restricted to its glycosylated form and occurred in three different regions. In addition, mass spectrometry and mutagenesis analyses both indicated that the major cleavage site laid in the C-terminal of the third transmembrane domain of TSAP6. A somatic cell knock-in approach was used to genetically inactivate the endogenous RHBDD1 in HCT116 and RKO colon cancer cells. Exosome secretion was significantly elevated when RHBDD1 was inactivated in the two cells lines. The increased exosome secretion was verfied through the detection of certain exosomal components, including Tsg101, Tf-R, FasL and Trail. In addition, the elevation of exosome secretion by RHBDD1 inactivation was reduced when TSAP6 was knocked down, indicating that the role of RHBDD1 in regulating exosomal trafficking is very likely to be TSAP6-dependent. We found that the increase in FasL and Trail increased exosome-induced apoptosis in Jurkat cells. Taken together, our findings suggest that RHBDD1 is involved in the regulation of a nonclassical exosomal secretion pathway through the restriction of TSAP6. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG
Protein markers
EV: TSG101/ Tf-receptor
non-EV: HSP90B1
Proteomics
no
EV density (g/ml)
1.06-1.11
Show all info
Study aim
Biogenesis/Sorting
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)
120
Density gradient
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Top-down
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101/ Tf-receptor
Detected contaminants
HSP90B1
ELISA
Detected EV-associated proteins
Tf-receptor
Characterization: Particle analysis
EV120041 1/1 Bos bovis Milk 0.2 µm filter
Density cushion
dUC
Sucrose-DG
Reinhardt TA 2012 56%

Study summary

Full title
All authors
Reinhardt TA, Lippolis JD, Nonnecke BJ, Sacco RE
Journal
J Proteomics
Abstract
Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in bio (show more...)Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology. (hide)
EV-METRIC
56% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Milk
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Density cushion + dUC + Sucrose-DG
Adj. k-factor
157.1 (pelleting)
Protein markers
EV: TSG101/ MFGE8
non-EV: None
Proteomics
yes
EV density (g/ml)
1.7
Show all info
Study aim
Omics
Sample
Species
Bos bovis
Sample Type
Milk
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)
60
Pelleting: rotor type
50.2Ti
Pelleting: adjusted k-factor
157.1
Density gradient
Lowest density fraction
5
Highest density fraction
43
Orientation
Bottom-up
Rotor type
50.2Ti
Speed (g)
200000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101/ MFGE8
ELISA
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV120038 2/2 Mus musculus Other 0.2 µm filter
dUC
Sucrose-DG
Perez-Gonzalez R 2012 56%

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
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 + Sucrose-DG
Protein markers
EV: Flotilin1/ ADAM10/ BACE1/ NISCASTRIN/ APP/ Beta-amyloid/ AChE
non-EV: None
Proteomics
no
EV density (g/ml)
1.07-1.17
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Mus musculus
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
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ ADAM10/ BACE1/ NISCASTRIN/ APP/ Beta-amyloid/ AChE
ELISA
Detected EV-associated proteins
ADAM10/ BACE1/ NISCASTRIN/ APP/ Beta-amyloid/ AChE
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120006 1/1 Homo sapiens Urine dUC Musante L 2012 56%

Study summary

Full title
All authors
Musante L, Saraswat M, Duriez E, Byrne B, Ravidà A, Domon B, Holthofer H
Journal
PLoS One
Abstract
Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, (show more...)Urinary exosomes represent a precious source of potential biomarkers for disease biology. Currently, the methods for vesicle isolation are severely restricted by the tendency of vesicle entrapment, e.g. by the abundant Tamm-Horsfall protein (THP) polymers. Treatment by reducing agents such as dithiothreitol (DTT) releases entrapped vesicles, thus increasing the final yield. However, this harsh treatment can cause remodelling of all those proteins which feature extra-vesicular domains stabilized by internal disulfide bridges and have detrimental effects on their biological activity. In order to optimize exosomal yield, we explore two vesicle treatment protocols - dithiothreitol (DTT) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic (CHAPS) - applied to the differential centrifugation protocol for exosomal vesicle isolation. The results show that CHAPS treatment does not affect vesicle morphology or exosomal marker distribution, thus eliminating most of THP interference. Moreover, the recovery and preservation of catalytic activity of two trans-membrane proteases, dipeptidyl peptidase IV and nephrilysin, was examined and found to be clearly superior after CHAPS treatment compared to DTT. Finally, proteomic profiling by mass spectrometry (MS) revealed that 76.2% of proteins recovered by CHAPS are common to those seen for DTT treatment, which illustrates underlining similarities between the two approaches. In conclusion, we provide a major improvement to currently-utilized urinary vesicle isolation strategies to allow recovery of urinary vesicles without the deleterious interference of abundant urinary proteins, while preserving typical protein folding and, consequently, the precious biological activity of urinary proteins which serve as valuable biomarkers. (hide)
EV-METRIC
56% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Focus vesicles
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
Adj. k-factor
78.45 (pelleting) / 78.45 (washing)
Protein markers
EV: CD63/ TSG101/ MFGE8
non-EV: Tamm-Horsfall glycoproteinNephrin
Proteomics
yes
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
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
78.45
Wash: volume per pellet (ml)
5
Wash: Rotor Type
70Ti
Wash: adjusted k-factor
78.45
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ MFGE8
Detected contaminants
Tamm-Horsfall glycoproteinNephrin
ELISA
Detected EV-associated proteins
MFGE8
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120022 1/2 Homo sapiens Other dUC
Sucrose-DG (valid.)
Kshirsagar SK 2012 56%

Study summary

Full title
All authors
Kshirsagar SK, Alam SM, Jasti S, Hodes H, Nauser T, Gilliam M, Billstrand C, Hunt JS, Petroff MG
Journal
Placenta
Abstract
The semiallogenic fetus is tolerated by the maternal immune system through control of innate and ada (show more...)The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular co-localization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 h. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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
dUC + Sucrose-DG (valid.)
Protein markers
EV: CD63/ LAMP1/ MHC1/ B7H-1/ B7H-3
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.15-1.18
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)
90
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Rotor type
TLA100.4
Speed (g)
110000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ LAMP1/ MHC1/ B7H-1/ B7H-3
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
LAMP1/ MHC1/ B7H-1/ B7H-3
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120031 2/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Iodixanol-DG (valid.)
UF
Epple LM 2012 56%

Study summary

Full title
All authors
Epple LM, Griffiths SG, Dechkovskaia AM, Dusto NL, White J, Ouellette RJ, Anchordoquy TJ, Bemis LT, Graner MW
Journal
PLoS One
Abstract
Medulloblastomas are the most prevalent malignant pediatric brain tumors. Survival for these patient (show more...)Medulloblastomas are the most prevalent malignant pediatric brain tumors. Survival for these patients has remained largely the same for approximately 20 years, and our therapies for these cancers cause significant health, cognitive, behavioral and developmental sequelae for those who survive the tumor and their treatments. We obviously need a better understanding of the biology of these tumors, particularly with regard to their migratory/invasive behaviors, their proliferative propensity, and their abilities to deflect immune responses. Exosomes, virus-sized membrane vesicles released extracellularly from cells after formation in, and transit thru, the endosomal pathway, may play roles in medulloblastoma pathogenesis but are as yet unstudied in this disease. Here we characterized exosomes from a medulloblastoma cell line with biochemical and proteomic analyses, and included characterization of patient serum exosomes. Further scrutiny of the proteomic data suggested functional properties of the exosomes that are relevant to medulloblastoma tumor biology, including their roles as proliferation stimulants, their activities as attractants for tumor cell migration, and their immune modulatory impacts on lymphocytes. Aspects of this held true for exosomes from other medulloblastoma cell lines as well. Additionally, pathway analyses suggested a possible role for the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A); however, inhibition of the protein's activity actually increased D283MED cell proliferation/clonogenecity, suggesting that HNF4A may act as a tumor suppressor in this cell line. Our work demonstrates that relevant functional properties of exosomes may be derived from appropriate proteomic analyses, which translate into mechanisms of tumor pathophysiology harbored in these extracellular vesicles. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes / extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Iodixanol-DG (valid.) + UF
Adj. k-factor
122.2 (pelleting) / 122.2 (washing)
Protein markers
EV: CD9/ HSP90/ HSP70/ AChE/ HSP60/ HSC70/ HSP27/ GAPDH
non-EV: None
Proteomics
yes
EV density (g/ml)
1.11-1.15
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
70.1Ti
Pelleting: adjusted k-factor
122.2
Wash: volume per pellet (ml)
8
Wash: Rotor Type
70.1Ti
Wash: adjusted k-factor
122.2
Density gradient
Only used for validation of main results
1
Highest density fraction
60
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ HSP90/ HSP70/ AChE/ HSP60/ HSC70/ HSP27/ GAPDH
ELISA
Detected EV-associated proteins
AChE/ HSP60/ HSC70/ HSP27/ GAPDH
Characterization: Particle analysis
DLS
NTA
EM
EM-type
transmission EM
Image type
Close-up
EV120015 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
Dreux M 2012 56%

Study summary

Full title
All authors
Dreux M, Garaigorta U, Boyd B, Décembre E, Chung J, Whitten-Bauer C, Wieland S, Chisari FV
Journal
Cell Host Microbe
Abstract
Viral nucleic acids often trigger an innate immune response in infected cells. Many viruses, includi (show more...)Viral nucleic acids often trigger an innate immune response in infected cells. Many viruses, including hepatitis C virus (HCV), have evolved mechanisms to evade intracellular recognition. Nevertheless, HCV-permissive cells can trigger a viral RNA-, TLR7-, and cell-contact-dependent compensatory interferon response in nonpermissive plasmacytoid dendritic cells (pDCs). Here we report that these events are mediated by transfer of HCV-RNA-containing exosomes from infected cells to pDCs. The exosomal viral RNA transfer is dependent on the endosomal sorting complex required for transport (ESCRT) machinery and on Annexin A2, an RNA-binding protein involved in membrane vesicle trafficking, and is suppressed by exosome release inhibitors. Further, purified concentrated HCV-RNA-containing exosomes are sufficient to activate pDCs. Thus, vesicular sequestration and exosomal export of viral RNA may serve both as a viral strategy to evade pathogen sensing within infected cells and as a host strategy to induce an unopposed innate response in replication-nonpermissive bystander cells. (hide)
EV-METRIC
56% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Adj. k-factor
230.8 (pelleting)
Protein markers
EV: CD63/ CD81
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.1-1.177
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)
120
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
230.8
Density gradient
Only used for validation of main results
1
Highest density fraction
2
Orientation
Top-down
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EV120024 3/3 Homo sapiens Cell culture supernatant 0.1 µm filter
Iodixanol-DG (valid.)
Tauro BJ 2012 50%

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
50% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 + Iodixanol-DG (valid.)
Protein markers
EV: Alix/ HSP70/ TSG101
non-EV: None
Proteomics
yes
EV density (g/ml)
1.110
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
Density gradient
Only used for validation of main results
1
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Speed (g)
100000
Pelleting-wash: volume per pellet (mL)
2
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
EV120014 1/1 Homo sapiens Cell culture supernatant Density cushion
Sucrose-DG
UF
Choi DS 2012 50%

Study summary

Full title
All authors
Choi DS, Yang JS, Choi EJ, Jang SC, Park S, Kim OY, Hwang D, Kim KP, Kim YK, Kim S, Gho YS
Journal
J Proteome Res
Abstract
Various mammalian cells including tumor cells secrete extracellular vesicles (EVs), otherwise known (show more...)Various mammalian cells including tumor cells secrete extracellular vesicles (EVs), otherwise known as exosomes and microvesicles. EVs are nanosized bilayered proteolipids and play multiple roles in intercellular communication. Although many vesicular proteins have been identified, their functional interrelationships and the mechanisms of EV biogenesis remain unknown. By interrogating proteomic data using systems approaches, we have created a protein interaction network of human colorectal cancer cell-derived EVs which comprises 1491 interactions between 957 vesicular proteins. We discovered that EVs have well-connected clusters with several hub proteins similar to other subcellular networks. We also experimentally validated that direct protein interactions between cellular proteins may be involved in protein sorting during EV formation. Moreover, physically and functionally interconnected protein complexes form functional modules involved in EV biogenesis and functions. Specifically, we discovered that SRC signaling plays a major role in EV biogenesis, and confirmed that inhibition of SRC kinase decreased the intracellular biogenesis and cell surface release of EVs. Our study provides global insights into the cargo-sorting, biogenesis, and pathophysiological roles of these complex extracellular organelles. (hide)
EV-METRIC
50% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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
Density cushion + Sucrose-DG + UF
Protein markers
EV: Alix/ CD63/ CD81/ CD9/ TSG101
non-EV: Cell organelle protein
Proteomics
yes
EV density (g/ml)
1.160
Show all info
Study aim
Omics
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
Density gradient
Lowest density fraction
0.6
Highest density fraction
2.8
Orientation
Bottom-up
Speed (g)
150000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD81/ CD9/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EV120013 1/1 Homo sapiens Cell culture supernatant Density cushion
Iodixanol-DG
UF
Choi DS 2012 50%

Study summary

Full title
All authors
Choi DS, Choi DY, Hong BS, Jang SC, Kim DK, Lee J, Kim YK, Kim KP, Gho YS
Journal
J Extracell Vesicles
Abstract
Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, in (show more...)Cancer cells actively release extracellular vesicles (EVs), including exosomes and microvesicles, into surrounding tissues. These EVs play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteomic differences between primary and metastatic cancer cell-derived EVs remain unclear. Here, we conducted comparative proteomic analysis between EVs derived from human primary colorectal cancer cells (SW480) and their metastatic derivatives (SW620). Using label-free quantitation, we identified 803 and 787 proteins in SW480 EVs and SW620 EVs, respectively. Based on comparison between the estimated abundance of EV proteins, we identified 368 SW480 EV-enriched and 359 SW620 EV-enriched proteins. SW480 EV-enriched proteins played a role in cell adhesion, but SW620 EV-enriched proteins were associated with cancer progression and functioned as diagnostic indicators of metastatic cancer; they were overexpressed in metastatic colorectal cancer and played roles in multidrug resistance. As the first proteomic analysis comparing primary and metastatic cancer-derived EVs, this study increases our understanding of the pathological function of EVs in the metastatic process and provides useful biomarkers for cancer metastasis. (hide)
EV-METRIC
50% (93rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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
Density cushion + Iodixanol-DG + UF
Protein markers
EV: Alix/ CD63/ CD81/ CD9/ LAMP1/ ICAM1/ Beta-actin
non-EV: None
Proteomics
yes
EV density (g/ml)
1.08-1.1
Show all info
Study aim
Omics
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
Density gradient
Lowest density fraction
5
Highest density fraction
30
Orientation
Bottom-up
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD81/ CD9/ LAMP1/ ICAM1/ Beta-actin
ELISA
Detected EV-associated proteins
LAMP1/ ICAM1/ Beta-actin
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV120103 1/2 Homo sapiens Serum dUC
Sucrose-DG (valid.)
de Hoog VC 2012 44%

Study summary

Full title
All authors
de Hoog VC, Timmers L, Schoneveld AH, Wang JW, van de Weg SM, Sze SK, van Keulen JK, Hoes AW, den Ruijter HM, de Kleijn DP, Mosterd A
Journal
Eur Heart J Acute Cardiovasc Care
Abstract
AIMS: Biomarkers are essential in the early detection of acute coronary syndromes (ACS). Serum extra (show more...)AIMS: Biomarkers are essential in the early detection of acute coronary syndromes (ACS). Serum extracellular vesicles are small vesicles in the plasma containing protein and RNA and have been shown to be involved in ACS-related processes like apoptosis and coagulation. Therefore, we hypothesized that serum extracellular vesicle protein levels are associated with ACS. METHODS AND RESULTS: Three serum extracellular vesicle proteins potentially associated with ACS were identified with differential Q-proteomics and were evaluated in 471 frozen serum samples of ACS-suspected patients presenting to the emergency department (30% of whom had an ACS). Protein levels were measured after vesicle isolation using ExoQuick. Mean serum extracellular vesicle concentration of the different proteins was compared between ACS and non-ACS patients. Selected proteins were tested in a univariate logistic regression model, as well as in a multivariate model to adjust for cardiovascular risk factors. A separate analysis was performed in men and women. In the multivariate logistic regression analysis, polygenic immunoglobulin receptor, (pIgR; OR 1.630, p=0.026), cystatin C (OR 1.641, p=0.021), and complement factor C5a (C5a, OR 1.495, p=0.025) were significantly associated with ACS, while total vesicle protein concentration was borderline significant. The association of the individual proteins with ACS was markedly stronger in men. CONCLUSIONS: These data show that serum extracellular vesicle pIgR, cystatin C, and C5a concentrations are independently associated with ACS and that there are pronounced gender differences. These observations should be validated in a large, prospective study to assess the potential role of vesicle content in the evaluation of patients suspected of having an ACS. (hide)
EV-METRIC
44% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: CD9
non-EV: None
Proteomics
yes
EV density (g/ml)
1.15-1.19
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Serum
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)
60
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.4
Highest density fraction
2.5
Orientation
Bottom-up
Rotor type
SW60
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9
Characterization: Particle analysis
EV120099 2/2 Mus musculus Cell culture supernatant dUC
Sucrose-DG (valid.)
Yuyama K 2012 44%

Study summary

Full title
All authors
Yuyama K, Sun H, Mitsutake S, Igarashi Y
Journal
J Biol Chem
Abstract
Amyloid ?-peptide (A?), the pathogenic agent of Alzheimer disease, is a physiological metabolite who (show more...)Amyloid ?-peptide (A?), the pathogenic agent of Alzheimer disease, is a physiological metabolite whose levels are constantly controlled in normal brain. Recent studies have demonstrated that a fraction of extracellular A? is associated with exosomes, small membrane vesicles of endosomal origin, although the fate of A? in association with exosome is largely unknown. In this study, we identified novel roles for neuron-derived exosomes acting on extracellular A?, i.e. exosomes drive conformational changes in A? to form nontoxic amyloid fibrils and promote uptake of A? by microglia. The A? internalized together with exosomes was further transported to lysosomes and degraded. We also found that blockade of phosphatidylserine on the surface of exosomes by annexin V not only prevented exosome uptake but also suppressed A? incorporation into microglia. In addition, we demonstrated that secretion of neuron-derived exosomes was modulated by the activities of sphingolipid-metabolizing enzymes, including neutral sphingomyelinase 2 (nSMase2) and sphingomyelin synthase 2 (SMS2). In transwell experiments, up-regulation of exosome secretion from neuronal cells by treatment with SMS2 siRNA enhanced A? uptake into microglial cells and significantly decreased extracellular levels of A?. Our findings indicate a novel mechanism responsible for clearance of A? through its association with exosomes. The modulation of the vesicle release and/or elimination may alter the risk of AD. (hide)
EV-METRIC
44% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: Alix/ TSG101/ GM1
non-EV: Cell organelle protein
Proteomics
no
EV density (g/ml)
1.12-1.16
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
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
2.2999999999999998
Orientation
Top-down
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ GM1
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
GM1
Characterization: Particle analysis
EV120044 1/1 Homo sapiens "Cerebrospinal fluid" dUC
Sucrose-DG
Street JM 2012 44%

Study summary

Full title
All authors
Street JM, Barran PE, Mackay CL, Weidt S, Balmforth C, Walsh TS, Chalmers RT, Webb DJ, Dear JW
Journal
J Transl Med
Abstract
BACKGROUND: Exosomes are released from multiple cell types, contain protein and RNA species, and hav (show more...)BACKGROUND: Exosomes are released from multiple cell types, contain protein and RNA species, and have been exploited as a novel reservoir for disease biomarker discovery. They can transfer information between cells and may cause pathology, for example, a role for exosomes has been proposed in the pathophysiology of Alzheimer's disease. Although studied in several biofluids, exosomes have not been extensively studied in the cerebrospinal fluid (CSF) from humans. The objective of this study was to determine: 1) whether human CSF contains exosomes and 2) the variability in exosomal protein content across individuals. METHODS: CSF was collected from 5 study participants undergoing thoraco-abdominal aortic aneurysm repair (around 200 - 500 ml per participant) and low-density membrane vesicles were concentrated by ultracentrifugation. The presence of exosomes was determined by western blot for marker proteins, isopycnic centrifugation on a sucrose step gradient and transmission electron microscopy with immuno-labelling. Whole protein profiling was performed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR). RESULTS: Flotillin 1 and tumor susceptibility gene 101 (TSG101), two exosomal marker proteins, were identified in the ultracentrifugation pellet using western blot. These markers localized to a density consistent with exosomes following isopycnic centrifugation. Transmission electron microscopy visualized structures consistent with exosomes in size and appearance that labelled positive for flotillin 1. Therefore, the pellet that resulted from ultracentrifugation of human CSF contained exosomes. FT-ICR profiling of this pellet was performed and 84-161 ions were detected per study participant. Around one third of these ions were only present in a single study participant and one third were detected in all five. With regard to ion quantity, the median coefficient of variation was 81% for ions detected in two or more samples. CONCLUSIONS: Exosomes were identified in human CSF and their proteome is a potential new reservoir for biomarker discovery in neurological disorders such as Alzheimer's disease. However, techniques used to concentrate exosomes from CSF need refinement to reduce variability. In this study we used relatively large starting volumes of human CSF, future studies will focus on exosome isolation from smaller real life clinical samples; a key challenge in the development of exosomes as translational tools. (hide)
EV-METRIC
44% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
"Cerebrospinal fluid"
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Protein markers
EV: Flotilin1/ TSG101
non-EV: None
Proteomics
yes
EV density (g/ml)
1.1-1.14
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
"Cerebrospinal fluid"
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
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ TSG101
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV120085 1/1 Homo sapiens Other 0.2 µm filter
dUC
SEC
Sucrose-DG
Ronquist GK 2012 44%

Study summary

Full title
All authors
Ronquist GK, Larsson A, Stavreus-Evers A, Ronquist G
Journal
Prostate
Abstract
BACKGROUND: Prostate acinar epithelial cells release microvesicles (prostasomes) that possess pleiot (show more...)BACKGROUND: Prostate acinar epithelial cells release microvesicles (prostasomes) that possess pleiotropic biological effects relevant for successful fertilization. Prostasomes are formed in a similar way as exosomes but are heterogeneous as regards size and appearance. Like exosomes they are thought to be mediators of intercellular communication. METHODS: We prepared seminal prostasomes in accordance with the prevailing protocol for exosome preparation including passage through a 0.2 µm filter and centrifugation in a sucrose gradient. RESULTS: We compared the filterable prostasomes with those trapped on the filter (nonfilterable prostasomes) and, qualitatively, no conspicuous differences were apparent regarding ultrastructure and SDS-PAGE banding pattern. Moreover, both types of prostasomes contained DNA fragments and Western blot revealed presence of prostate specific membrane antigen (PSMA), CD38, and annexin A1. CONCLUSIONS: Reasonably, prostasomes could be included in the exosome family and be regarded as one entity containing chromosomal DNA. (hide)
EV-METRIC
44% (85th 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 / 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
0.2 µm filter + dUC + SEC + Sucrose-DG
Adj. k-factor
126 (pelleting)
Protein markers
EV: Annexin1/ PSMA/ CD38
non-EV: None
Proteomics
no
EV density (g/ml)
1.13-1.26
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
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
90Ti
Pelleting: adjusted k-factor
126.0
Density gradient
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Top-down
Rotor type
90Ti
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Annexin1/ PSMA/ CD38
ELISA
Detected EV-associated proteins
Annexin1/ PSMA/ CD38
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120039 1/1 Homo sapiens Urine 0.2 µm filter
dUC
Sucrose-DG
Ramirez-Alvarado M 2012 44%

Study summary

Full title
All authors
Ramirez-Alvarado M, Ward CJ, Huang BQ, Gong X, Hogan MC, Madden BJ, Charlesworth MC, Leung N
Journal
PLoS One
Abstract
Renal involvement is a frequent consequence of plasma cell dyscrasias. The most common entities are (show more...)Renal involvement is a frequent consequence of plasma cell dyscrasias. The most common entities are light chain amyloidosis, monoclonal immunoglobulin deposition disease and myeloma cast nephropathy. Despite a common origin, each condition has its own unique histologic and pathophysiologic characteristic which requires a renal biopsy to distinguish. Recent studies have shown urinary exosomes containing kidney-derived membrane and cytosolic proteins that can be used to probe the proteomics of the entire urinary system from the glomerulus to the bladder. In this study, we analyzed urine exosomes to determine the differences between exosomes from patients with light chain amyloidosis, multiple myeloma, monoclonal gammopathy of undetermined significance, and non-paraproteinemia related kidney disease controls. In patients with light chain amyloidosis, multiple myeloma and monoclonal gammopathy of undetermined significance, immunoreactive proteins corresponding to monomeric light chains were found in exosomes by western blot. In all of the amyloidosis samples with active disease, high molecular weight immunoreactive species corresponding to a decamer were found which were not found in exosomes from the other diseases or in amyloidosis exosomes from patients in remission. Few or no light chains monomeric bands were found in non-paraproteinemia related kidney disease controls. Our results showed that urinary exosomes may have tremendous potential in furthering our understanding of the pathophysiology and diagnosis of plasma cell dyscrasia related kidney diseases. (hide)
EV-METRIC
44% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG
Adj. k-factor
134 (pelleting)
Protein markers
EV: Podocin
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
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
T647.5
Pelleting: adjusted k-factor
134.0
Density gradient
Lowest density fraction
5
Highest density fraction
30
Orientation
Top-down
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Podocin
ELISA
Detected EV-associated proteins
Podocin
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Close-up, Wide-field
EV120019 1/3 Homo sapiens Cell culture supernatant Density cushion (valid.)
dUC
Peinado H 2012 44%

Study summary

Full title
All authors
Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, Hergueta-Redondo M, Williams C, García-Santos G, Ghajar C, Nitadori-Hoshino A, Hoffman C, Badal K, Garcia BA, Callahan MK, Yuan J, Martins VR, Skog J, Kaplan RN, Brady MS, Wolchok JD, Chapman PB, Kang Y, Bromberg J, Lyden D
Journal
Nat Med
Abstract
Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma (show more...)Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process. (hide)
EV-METRIC
44% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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 (valid.) + dUC
Adj. k-factor
266.3 (pelleting) / 266.3 (washing)
Protein markers
EV: Alix/ TSG101/ HSC70/ GAPDH/ Phospho-ERK/ PhosphoS6-Kinase/ Met/ Phospho-MET/ Melan-A/ Tyrp2
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential 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
SureSpin630
Pelleting: adjusted k-factor
266.3
Wash: volume per pellet (ml)
20
Wash: Rotor Type
SureSpin630
Wash: adjusted k-factor
266.3
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ TSG101/ HSC70/ GAPDH/ Phospho-ERK/ PhosphoS6-Kinase/ Met/ Phospho-MET/ Melan-A/ Tyrp2
ELISA
Detected EV-associated proteins
HSC70/ GAPDH/ Phospho-ERK/ PhosphoS6-Kinase/ Met/ Phospho-MET/ Melan-A/ Tyrp2
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV120019 2/3 Homo sapiens Blood plasma 0.2 µm filter
Density cushion (valid.)
dUC
Peinado H 2012 44%

Study summary

Full title
All authors
Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, Hergueta-Redondo M, Williams C, García-Santos G, Ghajar C, Nitadori-Hoshino A, Hoffman C, Badal K, Garcia BA, Callahan MK, Yuan J, Martins VR, Skog J, Kaplan RN, Brady MS, Wolchok JD, Chapman PB, Kang Y, Bromberg J, Lyden D
Journal
Nat Med
Abstract
Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma (show more...)Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process. (hide)
EV-METRIC
44% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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 + Density cushion (valid.) + dUC
Adj. k-factor
266.3 (pelleting) / 266.3 (washing)
Protein markers
EV: HSP90/ HSP70/ Tyrp2/ VLA4
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
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
Pelleting: rotor type
SureSpin630
Pelleting: adjusted k-factor
266.3
Wash: volume per pellet (ml)
20
Wash: Rotor Type
SureSpin630
Wash: adjusted k-factor
266.3
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
HSP90/ HSP70/ Tyrp2/ VLA4
ELISA
Detected EV-associated proteins
Tyrp2/ VLA4
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up
EV120019 3/3 Homo sapiens Blood plasma 0.2 µm filter
dUC
Peinado H 2012 44%

Study summary

Full title
All authors
Peinado H, Aleckovic M, Lavotshkin S, Matei I, Costa-Silva B, Moreno-Bueno G, Hergueta-Redondo M, Williams C, García-Santos G, Ghajar C, Nitadori-Hoshino A, Hoffman C, Badal K, Garcia BA, Callahan MK, Yuan J, Martins VR, Skog J, Kaplan RN, Brady MS, Wolchok JD, Chapman PB, Kang Y, Bromberg J, Lyden D
Journal
Nat Med
Abstract
Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma (show more...)Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process. (hide)
EV-METRIC
44% (92nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
76.3 (pelleting) / 76.3 (washing)
Protein markers
EV: HSP90/ HSP70/ Tyrp2/ VLA4
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
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
Pelleting: rotor type
S100AT5
Pelleting: adjusted k-factor
76.3
Wash: Rotor Type
S100AT5
Wash: adjusted k-factor
76.3
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
HSP90/ HSP70/ Tyrp2/ VLA4
ELISA
Detected EV-associated proteins
Tyrp2/ VLA4
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up
EV120036 1/1 Homo sapiens Cell culture supernatant dUC Palazzolo G 2012 44%

Study summary

Full title
All authors
Palazzolo G, Albanese NN, DI Cara G, Gygax D, Vittorelli ML, Pucci-Minafra I
Journal
Anticancer Res
Abstract
BACKGROUND/AIM: The phenomenon of membrane vesicle-release by neoplastic cells is a growing field of (show more...)BACKGROUND/AIM: The phenomenon of membrane vesicle-release by neoplastic cells is a growing field of interest in cancer research, due to their potential role in carrying a large array of tumor antigens when secreted into the extracellular medium. In particular, experimental evidence show that at least some of the tumor markers detected in the blood circulation of mammary carcinoma patients are carried by membrane-bound vesicles. Thus, biomarker research in breast cancer can gain great benefits from vesicle characterization. MATERIALS AND METHODS: Conditioned medium was collected from serum starved MDA-MB-231 sub-confluent cell cultures and exosome-like vesicles (ELVs) were isolated by ultracentrifugation. Ultrastructural analysis of ELVs was performed by transmission electron microscopy (TEM) and the purity of fraction was confirmed by western blotting assays. Proteomic profile of ELVs was carried out by 2 D-PAGE and protein identification performed by MALDI-ToF Mass Spectrometry. RESULTS: On the basis of ultrastructural and immunological characterization, the isolated vesicles have been classified as exosome-like vesicles (ELVs). The proteomic investigation showed a distinctive protein profile of the ELVs, in comparison to the whole cell lisates (WCL) proteome, which could be instrumental for cancer progression. The proteins were clustered into functional categories, according to the current bioinformatics resources and a Venn diagram was constructed based on these clusters. CONCLUSION: It is reasonable to assume that vesicle production allows neoplastic cells to exert different effects, according to the possible acceptor targets. For instance, vesicles could potentiate the malignant properties of adjacent neoplastic cells or activate non-tumoral cells. Moreover, vesicles could convey signals to immune cells and surrounding stroma cells. The present study may significantly contribute to the knowledge of the vesiculation phenomenon, which is a critical device for trans cellular communication in cancer. (hide)
EV-METRIC
44% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
Exosome-like vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
159.6 (pelleting)
Protein markers
EV: HSC70/ LAMP1
non-EV: Cell organelle protein
Proteomics
yes
TEM measurements
40-80
Show all info
Study aim
Omics
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)
90
Pelleting: rotor type
60Ti
Pelleting: adjusted k-factor
159.6
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
HSC70/ LAMP1
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
HSC70/ LAMP1
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120050 2/2 Mus musculus Other dUC
Sucrose-DG
Nolte-'t Hoen EN 2012 44%

Study summary

Full title
All authors
Nolte-'t Hoen EN, van der Vlist EJ, Aalberts M, Mertens HC, Bosch BJ, Bartelink W, Mastrobattista E, van Gaal EV, Stoorvogel W, Arkesteijn GJ, Wauben MH
Journal
Nanomedicine
Abstract
Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and hig (show more...)Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications.FROM THE CLINICAL EDITOR: The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology. (hide)
EV-METRIC
44% (85th 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
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 + Sucrose-DG
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: CD9
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.18
Show all info
Study aim
Technical
Sample
Species
Mus musculus
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
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
253.9
Density gradient
Lowest density fraction
0.4
Highest density fraction
2
Orientation
Bottom-up
Rotor type
SW60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9
Characterization: Particle analysis
Particle analysis: flow cytometry
EV120034 1/2 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
Luga V 2012 44%

Study summary

Full title
All authors
Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, Buchanan M, Hosein AN, Basik M, Wrana JL
Journal
Cell
Abstract
Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promote (show more...)Stroma in the tumor microenvironment plays a critical role in cancer progression, but how it promotes metastasis is poorly understood. Exosomes are small vesicles secreted by many cell types and enable a potent mode of intercellular communication. Here, we report that fibroblast-secreted exosomes promote breast cancer cell (BCC) protrusive activity and motility via Wnt-planar cell polarity (PCP) signaling. We show that exosome-stimulated BCC protrusions display mutually exclusive localization of the core PCP complexes, Fzd-Dvl and Vangl-Pk. In orthotopic mouse models of breast cancer, coinjection of BCCs with fibroblasts dramatically enhances metastasis that is dependent on PCP signaling in BCCs and the exosome component, Cd81 in fibroblasts. Moreover, we demonstrate that trafficking in BCCs promotes tethering of autocrine Wnt11 to fibroblast-derived exosomes. This work reveals an intercellular communication pathway whereby fibroblast exosomes mobilize autocrine Wnt-PCP signaling to drive BCC invasive behavior. (hide)
EV-METRIC
44% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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: CD81/ Flotilin1/ Actin/ Tubulin
non-EV: None
Proteomics
yes
EV density (g/ml)
1.13-1.15
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)
120-840
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD81/ Flotilin1/ Actin/ Tubulin
ELISA
Detected EV-associated proteins
Actin/ Tubulin
Characterization: Particle analysis
EV120005 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Lässer C 2012 44%

Study summary

Full title
All authors
Lässer C, Eldh M, Lötvall J
Journal
J Vis Exp
Abstract
The field of exosome research is rapidly expanding, with a dramatic increase in publications in rece (show more...)The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen, eradication of established tumors in mice, inhibition and activation of natural killer cells, promotion of differentiation into T regulatory cells, stimulation of T cell proliferation and induction of T cell apoptosis. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer. (hide)
EV-METRIC
44% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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/ HSP70/ Flotillin/ MHC2
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential 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/ CD81/ CD9/ HSP70/ Flotillin/ MHC2
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Flotillin/ MHC2
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120032 1/1 Homo sapiens Cell culture supernatant dUC
Sucrose-DG (valid.)
Kiss K 2012 44%

Study summary

Full title
All authors
Kiss K, Brozik A, Kucsma N, Toth A, Gera M, Berry L, Vallentin A, Vial H, Vidal M, Szakacs G
Journal
PLoS One
Abstract
ABCB6, a member of the adenosine triphosphate-binding cassette (ABC) transporter family, has been pr (show more...)ABCB6, a member of the adenosine triphosphate-binding cassette (ABC) transporter family, has been proposed to be responsible for the mitochondrial uptake of porphyrins. Here we show that ABCB6 is a glycoprotein present in the membrane of mature erythrocytes and in exosomes released from reticulocytes during the final steps of erythroid maturation. Consistent with its presence in exosomes, endogenous ABCB6 is localized to the endo/lysosomal compartment, and is absent from the mitochondria of cells. Knock-down studies demonstrate that ABCB6 function is not required for de novo heme biosynthesis in differentiating K562 cells, excluding this ABC transporter as a key regulator of porphyrin synthesis. We confirm the mitochondrial localization of ABCB7, ABCB8 and ABCB10, suggesting that only three ABC transporters should be classified as mitochondrial proteins. Taken together, our results challenge the current paradigm linking the expression and function of ABCB6 to mitochondria. (hide)
EV-METRIC
44% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: Alix/ Flotilin1/ TSG101/ Tf-receptor
non-EV: Spectrin
Proteomics
no
EV density (g/ml)
1.14-1.2
Show all info
Study aim
Other/(Mitochondrial) protein localization
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
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)
120
Density gradient
Only used for validation of main results
1
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Top-down
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Flotilin1/ TSG101/ Tf-receptor
Detected contaminants
Spectrin
ELISA
Detected EV-associated proteins
Tf-receptor
Characterization: Particle analysis
EV120017 1/1 Mus musculus Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG
Gennebäck N 2012 44%

Study summary

Full title
All authors
Gennebäck N, Hellman U, Malm L, Larsson G, Ronquist G, Waldenström A, Mörner S
Journal
J Extracell Vesicles
Abstract
Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or (show more...)Exosomes are nano-sized extracellular vesicles, released from various cells, which can stimulate or repress responses in targets cells. We recently reported that cultured cardiomyocytes are able to release exosomes and that they, in turn, are involved in facilitating events in target cells by alteration of gene expression. We investigated whether external stimuli of the cardiomyocyte might influence the transcriptional content of the released exosomes. Exosomes were isolated from media collected from cultured cardiomyocytes (HL-1) with or without growth factor treatment (TGF-?2 and PDGF-BB), with a series of differential centrifugations, including preparative ultracentrifugation and separation with a sucrose gradient. The exosomes were characterized with dynamic light scattering (DLS), electron microscopy (EM) and Western blot and analyzed with Illumina whole genome microarray gene expression. The exosomes were rounded in shape and had an average size of 50-90 nm in diameter with no difference between treatment groups. Analysis of the mRNA content in repeated experiments conclusively revealed 505 transcripts in the control group, 562 in the TGF-?2-treated group and 300 in the PDGF-BB-treated group. Common transcripts (217) were found in all 3 groups. We show that the mode of stimulation of parental cells affects the characteristics of exosomes released. Hence, there is a difference in mRNA content between exosomes derived from cultured cardiomyocytes stimulated, or not stimulated, with growth factors. We also conclude that all exosomes contain a basic package consisting of ribosomal transcripts and mRNAs coding for proteins with functions within the energy supply system. (hide)
EV-METRIC
44% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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
Adj. k-factor
255.8 (pelleting)
Protein markers
EV: CD63/ TSG101
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Omics
Sample
Species
Mus musculus
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)
120
Pelleting: rotor type
SW41
Pelleting: adjusted k-factor
255.8
Density gradient
Lowest density fraction
20
Highest density fraction
40
Orientation
Top-down
Rotor type
SW41
Speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
DLS
EM
EM-type
transmission EM
Image type
Close-up
EV120016 1/1 Homo sapiens
Mus musculus
Cell culture supernatant 0.2 µm filter
dUC
El-Andaloussi S 2012 44%

Study summary

Full title
All authors
El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C, Alvarez-Erviti L, Sargent IL, Wood MJ
Journal
Nat Protoc
Abstract
The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug (show more...)The use of small interfering RNAs (siRNAs) to induce gene silencing has opened a new avenue in drug discovery. However, their therapeutic potential is hampered by inadequate tissue-specific delivery. Exosomes are promising tools for drug delivery across different biological barriers. Here we show how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, we explain how to purify and characterize exosomes from transfected cell supernatant. Next, we detail crucial steps for loading siRNA into exosomes. Finally, we outline how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. Examples of anticipated results in which exosome-mediated siRNA delivery is evaluated by functional assays and imaging are also provided. The entire protocol takes ~3 weeks. (hide)
EV-METRIC
44% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
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: CD9/ Flotilin1/ LAMP1/ MHC2
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens / 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 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
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ Flotilin1/ LAMP1/ MHC2
ELISA
Detected EV-associated proteins
LAMP1/ MHC2
Characterization: Particle analysis
NTA
EV120010 3/6 Homo sapiens Urine dUC Alvarez ML 2012 44%

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
44% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
155.1 (pelleting) / 155.1 (washing)
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)
70
Pelleting: rotor type
SW32
Pelleting: adjusted k-factor
155.1
Wash: volume per pellet (ml)
25
Wash: Rotor Type
SW32
Wash: adjusted k-factor
155.1
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD9/ TSG101
ELISA
Detected EV-associated proteins
Alix/ CD9/ TSG101
Characterization: Particle analysis
EV120010 6/6 Homo sapiens Urine 0.2 µm filter
dUC
Alvarez ML 2012 44%

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
44% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
155.1 (pelleting) / 155.1 (washing)
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)
70
Pelleting: rotor type
SW32
Pelleting: adjusted k-factor
155.1
Wash: volume per pellet (ml)
25
Wash: Rotor Type
SW32
Wash: adjusted k-factor
155.1
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD9/ TSG101
ELISA
Detected EV-associated proteins
Alix/ CD9/ TSG101
Characterization: Particle analysis
EV120093 1/1 Mus musculus Cell culture supernatant dUC
Sucrose-DG
van der Vlist EJ 2012 43%

Study summary

Full title
All authors
van der Vlist EJ, Arkesteijn GJ, van de Lest CH, Stoorvogel W, Nolte-'t Hoen EN, Wauben MH
Journal
J Extracell Vesicles
Abstract
Many cell types release nanosized vesicles derived from endosomal compartments (exosomes) or the pla (show more...)Many cell types release nanosized vesicles derived from endosomal compartments (exosomes) or the plasma membrane. Vesicles actively released by CD4(+) T cells have immune-modulatory characteristics. Using our recently developed high-resolution flow cytometry-based method for the analysis of individual nanosized vesicles, we here investigated how T cell receptor (TCR)-triggering and co-stimulatory signals influence the quantity and characteristics of nanosized vesicles released by CD4(+) T cells. We found that the number of released nanosized vesicles within the buoyant density range characteristic for exosomes (1.10-1.19 g/ml) was increased by TCR-triggering and that additional co-stimulatory signals had a potentiating effect on vesicle release. However, the increase in the number of released vesicles varied substantially between density fractions within the 1.10-1.19 g/ml range and was highest for the vesicle populations in 1.14 and 1.17 g/ml fractions. Heterogeneity was also observed within the individual density fractions. Based on lipid bilayer fluorescent labelling intensity and light scattering, 3 distinct vesicle subpopulations were identified. One vesicle subpopulation increased significantly more upon T cell activation than the other subpopulations, and this was dependent on high levels of co-stimulation. These data show that T cells release a heterogeneous population of nanosized vesicles and indicate that T cells differentially regulate the release of distinct vesicle subpopulations depending on their activation status. (hide)
EV-METRIC
43% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
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 + Sucrose-DG
Adj. k-factor
276.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.1-1.19
Show all info
Study aim
Biogenesis/Sorting
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 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
276.6
Density gradient
Lowest density fraction
0.4
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Particle analysis
NTA
Particle analysis: flow cytometry
EV120023 2/3 Homo sapiens Cell culture supernatant 0.45 µm filter
dUC
Sucrose-DG
Palma J 2012 43%

Study summary

Full title
All authors
Palma J, Yaddanapudi SC, Pigati L, Havens MA, Jeong S, Weiner GA, Weimer KM, Stern B, Hastings ML, Duelli DM
Journal
Nucleic Acids Res
Abstract
MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previou (show more...)MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body. (hide)
EV-METRIC
43% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
Particles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + dUC + Sucrose-DG
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.13-1.19
TEM measurements
58-72
Show all info
Study aim
Biomarker
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 50,000 g and 100,000 g
Pelleting: time(min)
60
Density gradient
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV120023 3/3 Homo sapiens Milk 0.45 µm filter
dUC
Sucrose-DG
Palma J 2012 43%

Study summary

Full title
All authors
Palma J, Yaddanapudi SC, Pigati L, Havens MA, Jeong S, Weiner GA, Weimer KM, Stern B, Hastings ML, Duelli DM
Journal
Nucleic Acids Res
Abstract
MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previou (show more...)MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body. (hide)
EV-METRIC
43% (84th 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
Particles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.45 µm filter + dUC + Sucrose-DG
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.15;1.25
TEM measurements
130-180
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Milk
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Below or equal to 800 g
Between 50,000 g and 100,000 g
Pelleting: time(min)
60
Density gradient
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Speed (g)
100000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120050 1/2 Mus musculus Cell culture supernatant dUC
Sucrose-DG
Nolte-'t Hoen EN 2012 43%

Study summary

Full title
All authors
Nolte-'t Hoen EN, van der Vlist EJ, Aalberts M, Mertens HC, Bosch BJ, Bartelink W, Mastrobattista E, van Gaal EV, Stoorvogel W, Arkesteijn GJ, Wauben MH
Journal
Nanomedicine
Abstract
Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and hig (show more...)Nanosized cell-derived membrane vesicles are increasingly recognized as therapeutic vehicles and high-potential biomarkers for several diseases. Currently available methods allow bulk analysis of vesicles but are not suited for accurate quantification and fail to reveal phenotypic heterogeneity in membrane vesicle populations. For such analyses, single vesicle-based, multiparameter, high-throughput methods are needed. We developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized membrane vesicles. Proof of principle was obtained by single-particle analysis of virions and liposomes. Further validation was obtained by quantification of cell-derived nanosized membrane vesicles from cell cultures and body fluids. An important aspect was that the technology was extended to detect specific proteins on individual vesicles. This allowed identification of exosome subsets and phenotyping of individual exosomes produced by dendritic cells (DCs) undergoing different modes of activation. The described technology allows quantitative, multiparameter, and high-throughput analysis of a wide variety of nanosized particles and has broad applications.FROM THE CLINICAL EDITOR: The authors developed a fluorescence-based, high-resolution flow cytometric method for quantitative and qualitative analysis of nanosized cell-derived membrane vesicles that are increasingly recognized both as therapeutic vehicles and high-potential biomarkers for several diseases. A high throughput, easily available, and sensitive detection method such as the one discussed here is a critically important prerequisite for further refinements of this technology. (hide)
EV-METRIC
43% (82nd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Focus vesicles
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 + Sucrose-DG
Adj. k-factor
276.6 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.09-1.17
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
Between 50,000 g and 100,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
276.6
Density gradient
Lowest density fraction
0.4
Highest density fraction
2
Orientation
Bottom-up
Rotor type
SW60
Speed (g)
100000
Characterization: Particle analysis
NTA
Particle analysis: flow cytometry
EV120076 1/1 Echinostoma caproni
Fasciola hepatica
Other 0.2 µm filter
dUC
Marcilla A 2012 43%

Study summary

Full title
All authors
Marcilla A, Trelis M, Cortés A, Sotillo J, Cantalapiedra F, Minguez MT, Valero ML, Sánchez del Pino MM, Muñoz-Antoli C, Toledo R, Bernal D
Journal
PLoS One
Abstract
The study of host-parasite interactions has increased considerably in the last decades, with many st (show more...)The study of host-parasite interactions has increased considerably in the last decades, with many studies focusing on the identification of parasite molecules (i.e. surface or excretory/secretory proteins (ESP)) as potential targets for new specific treatments and/or diagnostic tools. In parallel, in the last few years there have been significant advances in the field of extracellular vesicles research. Among these vesicles, exosomes of endocytic origin, with a characteristic size ranging from 30-100 nm, carry several atypical secreted proteins in different organisms, including parasitic protozoa. Here, we present experimental evidence for the existence of exosome-like vesicles in parasitic helminths, specifically the trematodes Echinostoma caproni and Fasciola hepatica. These microvesicles are actively released by the parasites and are taken up by host cells. Trematode extracellular vesicles contain most of the proteins previously identified as components of ESP, as confirmed by proteomic, immunogold labeling and electron microscopy studies. In addition to parasitic proteins, we also identify host proteins in these structures. The existence of extracellular vesicles explains the secretion of atypical proteins in trematodes, and the demonstration of their uptake by host cells suggests an important role for these structures in host-parasite communication, as described for other infectious agents. (hide)
EV-METRIC
43% (81st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Other
Focus vesicles
extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Adj. k-factor
74.34 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Echinostoma caproni / Fasciola hepatica
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)
60
Pelleting: rotor type
TLA55
Pelleting: adjusted k-factor
74.34
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM/ scanning EM
Image type
Close-up, Wide-field