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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
EV120081 1/2 Homo sapiens Cell culture supernatant dUC Park JA 2012 22%

Study summary

Full title
All authors
Park JA, Sharif AS, Tschumperlin DJ, Lau L, Limbrey R, Howarth P, Drazen JM
Journal
J Allergy Clin Immunol
Abstract
BACKGROUND: Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role (show more...)BACKGROUND: Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role in angiogenesis. TF expression in the airways is associated with asthma, a disease characterized in part by subepithelial angiogenesis. OBJECTIVES: To determine potential sources of TF and the mechanisms of its availability in the lung microenvironment. METHODS: Normal human bronchial epithelial cells grown in air-liquid interface culture were subjected to a compressive stress of 30 cm H(2)O; this is comparable to that generated in the airway epithelium during bronchoconstriction in asthma. Conditioned media and cells were harvested to measure TF mRNA and TF protein. We also tested bronchoalveolar lavage fluid and airway biopsies from asthmatic patients and healthy controls for TF. RESULTS: TF mRNA was upregulated 2.2-fold after 3 hours of stress compared with unstressed cells. Intracellular and secreted TF proteins were enhanced 1.6-fold and more than 50-fold, respectively, compared with those of control cells after the onset of compression. The amount of TF in the bronchoalveolar lavage fluid from patients with asthma was found at mean concentrations that were 5 times greater than those of healthy controls. Immunohistochemical staining of endobronchial biopsies identified epithelial localization of TF with increased expression in asthma. Exosomes isolated from the conditioned media of normal human bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic subjects by ultracentrifugation contained TF. CONCLUSIONS: Our in vitro and in vivo studies show that mechanically stressed bronchial epithelial cells are a source of secreted TF and that exosomes are potentially a key carrier of the TF signal. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: Annexin5/ GAPDH/ Actin/ EGFR/ TF
non-EV: Cell organelle protein/ "E-cadherin/ Integrin-beta5"
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Annexin5/ GAPDH/ Actin/ EGFR/ TF
Detected contaminants
Cell organelle protein/ "E-cadherin/ Integrin-beta5"
ELISA
Detected EV-associated proteins
Annexin5/ GAPDH/ Actin/ EGFR/ TF
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120081 2/2 Homo sapiens BALF dUC Park JA 2012 22%

Study summary

Full title
All authors
Park JA, Sharif AS, Tschumperlin DJ, Lau L, Limbrey R, Howarth P, Drazen JM
Journal
J Allergy Clin Immunol
Abstract
BACKGROUND: Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role (show more...)BACKGROUND: Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role in angiogenesis. TF expression in the airways is associated with asthma, a disease characterized in part by subepithelial angiogenesis. OBJECTIVES: To determine potential sources of TF and the mechanisms of its availability in the lung microenvironment. METHODS: Normal human bronchial epithelial cells grown in air-liquid interface culture were subjected to a compressive stress of 30 cm H(2)O; this is comparable to that generated in the airway epithelium during bronchoconstriction in asthma. Conditioned media and cells were harvested to measure TF mRNA and TF protein. We also tested bronchoalveolar lavage fluid and airway biopsies from asthmatic patients and healthy controls for TF. RESULTS: TF mRNA was upregulated 2.2-fold after 3 hours of stress compared with unstressed cells. Intracellular and secreted TF proteins were enhanced 1.6-fold and more than 50-fold, respectively, compared with those of control cells after the onset of compression. The amount of TF in the bronchoalveolar lavage fluid from patients with asthma was found at mean concentrations that were 5 times greater than those of healthy controls. Immunohistochemical staining of endobronchial biopsies identified epithelial localization of TF with increased expression in asthma. Exosomes isolated from the conditioned media of normal human bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic subjects by ultracentrifugation contained TF. CONCLUSIONS: Our in vitro and in vivo studies show that mechanically stressed bronchial epithelial cells are a source of secreted TF and that exosomes are potentially a key carrier of the TF signal. (hide)
EV-METRIC
22% (70th 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
BALF
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: Annexin5/ GAPDH/ Actin/ EGFR/ TF
non-EV: Cell organelle protein/ "E-cadherin/ Integrin-beta5"
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
BALF
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Annexin5/ GAPDH/ Actin/ EGFR/ TF
Detected contaminants
Cell organelle protein/ "E-cadherin/ Integrin-beta5"
ELISA
Detected EV-associated proteins
Annexin5/ GAPDH/ Actin/ EGFR/ TF
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120106 3/4 Homo sapiens Cell culture supernatant dUC
Sucrose-DG
Maguire CA 2012 22%

Study summary

Full title
All authors
Maguire CA, Balaj L, Sivaraman S, Crommentuijn MH, Ericsson M, Mincheva-Nilsson L, Baranov V, Gianni D, Tannous BA, Sena-Esteves M, Breakefield XO, Skog J
Journal
Mol Ther
Abstract
Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured (show more...)Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG
Protein markers
EV: Alix
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
25
Density gradient
Density medium
Sucrose
Lowest density fraction
8
Highest density fraction
60
Orientation
Top-down
Rotor type
MLS50
Speed (g)
100000
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix
Characterization: Particle analysis
EV120144 1/1 Homo sapiens Cell culture supernatant dUC Li M 2012 22%

Study summary

Full title
All authors
Li M, Aliotta JM, Asara JM, Tucker L, Quesenberry P, Lally M, Ramratnam B
Journal
Proteomics
Abstract
HIV-1 infection causes profound effects both inside and outside of cells through multiple mechanisms (show more...)HIV-1 infection causes profound effects both inside and outside of cells through multiple mechanisms, including those mediated by exosomes. Using the technique of stable isotope labeling by amino acids in cell culture, we compared protein expression patterns in the exosomal compartment of HIV-1-infected and -uninfected lymphocytic H9 cells. Of 770 proteins identified in two independent sets of exosomal samples, 14 proteins were found to be differentially expressed in the exosomal fraction of HIV-1-infected cells versus -uninfected controls. Gene Ontology survey and DAVID analysis revealed that identified proteins were enriched for functional categories such as binding. Of these 14 proteins, three immunomodulatory molecules were reproducibly identified in both replicates and included ADP-ribosyl cyclase 1 (CD38), L-lactate dehydrogenase B chain (LDHB), and Annexin A5 (ANXA5). In addition to previously reported HIV-1 associations with CD38 and LDHB, new interactions were identified and validated for ANXA5, CD38, and LDHB, which were found to bind to HIV-1 p24 and Tat. In summary, our studies reveal that exosomes released from HIV-1-infected cells are composed of a unique and quantitatively different protein signature and harbor regulatory molecules that impact the processes of cellular apoptosis (ANXA5 and LDHB) and proliferation (CD38). (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
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
240.8 (pelleting)
Protein markers
EV: Annexin5
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
TH641
Pelleting: adjusted k-factor
240.8
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Annexin5
ELISA
Detected EV-associated proteins
Annexin5
Characterization: Particle analysis
EV120051 1/3 Mus musculus Cell culture supernatant dUC Lee C 2012 22%

Study summary

Full title
All authors
Lee C, Mitsialis SA, Aslam M, Vitali SH, Vergadi E, Konstantinou G, Sdrimas K, Fernandez-Gonzalez A, Kourembanas S
Journal
Circulation
Abstract
BACKGROUND: Hypoxia induces an inflammatory response in the lung manifested by alternative activatio (show more...)BACKGROUND: Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of proinflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension. Mesenchymal stromal cell transplantation inhibits lung inflammation, vascular remodeling, and right heart failure and reverses hypoxic pulmonary hypertension in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which mesenchymal stromal cells are protective in hypoxic pulmonary hypertension. METHODS AND RESULTS: We fractionated mouse mesenchymal stromal cell-conditioned media to identify the biologically active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of proinflammatory and proproliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of hypoxic pulmonary hypertension. Intravenous delivery of mesenchymal stromal cell-derived exosomes (MEX) inhibited vascular remodeling and hypoxic pulmonary hypertension, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA, the expression of which is decreased in human pulmonary hypertension. MEX produced by human umbilical cord mesenchymal stromal cells inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells, demonstrating a direct effect of MEX on hypoxic vascular cells. CONCLUSION: This study indicates that MEX exert a pleiotropic protective effect on the lung and inhibit pulmonary hypertension through suppression of hyperproliferative pathways, including STAT3-mediated signaling induced by hypoxia. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD63/ Flotilin1/ HSP90
non-EV: Dicer
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ HSP90
Detected contaminants
Dicer
Characterization: Particle analysis
EV120051 3/3 Mus musculus Cell culture supernatant 0.2 µm filter
dUC
SEC
UF
Lee C 2012 22%

Study summary

Full title
All authors
Lee C, Mitsialis SA, Aslam M, Vitali SH, Vergadi E, Konstantinou G, Sdrimas K, Fernandez-Gonzalez A, Kourembanas S
Journal
Circulation
Abstract
BACKGROUND: Hypoxia induces an inflammatory response in the lung manifested by alternative activatio (show more...)BACKGROUND: Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of proinflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension. Mesenchymal stromal cell transplantation inhibits lung inflammation, vascular remodeling, and right heart failure and reverses hypoxic pulmonary hypertension in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which mesenchymal stromal cells are protective in hypoxic pulmonary hypertension. METHODS AND RESULTS: We fractionated mouse mesenchymal stromal cell-conditioned media to identify the biologically active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of proinflammatory and proproliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of hypoxic pulmonary hypertension. Intravenous delivery of mesenchymal stromal cell-derived exosomes (MEX) inhibited vascular remodeling and hypoxic pulmonary hypertension, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA, the expression of which is decreased in human pulmonary hypertension. MEX produced by human umbilical cord mesenchymal stromal cells inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells, demonstrating a direct effect of MEX on hypoxic vascular cells. CONCLUSION: This study indicates that MEX exert a pleiotropic protective effect on the lung and inhibit pulmonary hypertension through suppression of hyperproliferative pathways, including STAT3-mediated signaling induced by hypoxia. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + SEC + UF
Protein markers
EV: Alix/ CD63/ CD81/ CD9/ Flotilin1/ HSP90/ TSG101
non-EV: Dicer
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ CD81/ CD9/ Flotilin1/ HSP90/ TSG101
Detected contaminants
Dicer
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120067 2/2 Homo sapiens Blood plasma 0.2 µm filter
dUC
Khan S 2012 22%

Study summary

Full title
All authors
Khan S, Jutzy JM, Valenzuela MM, Turay D, Aspe JR, Ashok A, Mirshahidi S, Mercola D, Lilly MB, Wall NR
Journal
PLoS One
Abstract
BACKGROUND: Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa ce (show more...)BACKGROUND: Survivin is expressed in prostate cancer (PCa), and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment. METHODS: Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively. RESULTS: Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six) or high (nine) Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls. CONCLUSIONS: These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection. (hide)
EV-METRIC
22% (60th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
DNF
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: LAMP1/ AChE
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
1080
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
LAMP1/ AChE
ELISA
Detected EV-associated proteins
LAMP1/ AChE
Characterization: Particle analysis
EV120066 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Hu G 2012 22%

Study summary

Full title
All authors
Hu G, Yao H, Chaudhuri AD, Duan M, Yelamanchili SV, Wen H, Cheney PD, Fox HS, Buch S
Journal
Cell Death Dis
Abstract
Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug (show more...)Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3'-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
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
110.3 (pelleting)
Protein markers
EV: TSG101
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
110.3
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
TSG101
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120065 1/2 Rattus norvegicus/rattus Cell culture supernatant dUC
UF
Hooper C 2012 22%

Study summary

Full title
All authors
Hooper C, Sainz-Fuertes R, Lynham S, Hye A, Killick R, Warley A, Bolondi C, Pocock J, Lovestone S
Journal
BMC Neurosci
Abstract
BACKGROUND: Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both pla (show more...)BACKGROUND: Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a. RESULTS: Wnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including ?-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia. CONCLUSION: These findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + UF
Protein markers
EV: Alix/ Beta-actin
non-EV: None
Proteomics
yes
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Beta-actin
ELISA
Detected EV-associated proteins
Beta-actin
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Wide-field
EV120065 2/2 Rattus norvegicus/rattus Cell culture supernatant dUC
UF
Hooper C 2012 22%

Study summary

Full title
All authors
Hooper C, Sainz-Fuertes R, Lynham S, Hye A, Killick R, Warley A, Bolondi C, Pocock J, Lovestone S
Journal
BMC Neurosci
Abstract
BACKGROUND: Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both pla (show more...)BACKGROUND: Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a. RESULTS: Wnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including ?-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia. CONCLUSION: These findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + UF
Protein markers
EV: Alix/ Beta-actin
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/Sorting
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ Beta-actin
ELISA
Detected EV-associated proteins
Beta-actin
Characterization: Particle analysis
EV120064 1/1 Homo sapiens Cell culture supernatant dUC Hessvik NP 2012 22%

Study summary

Full title
All authors
Hessvik NP, Phuyal S, Brech A, Sandvig K, Llorente A
Journal
Biochim Biophys Acta Gene Regul Mech
Abstract
Exosomes are small extracellular vesicles released to the extracellular milieu through fusion of mul (show more...)Exosomes are small extracellular vesicles released to the extracellular milieu through fusion of multivesicular bodies with the plasma membrane. These vesicles contain microRNAs and might therefore be vehicles transferring genetic information between cells. The aim of this study was to investigate whether there was a sorting of microRNAs into exosomes in the prostate cancer cell line PC-3. In addition, microRNAs in PC-3 cells and in the non-cancerous prostate cell line RWPE-1 were compared. Exosomes were isolated from the conditioned media from PC-3 cells by ultracentrifugation and inspected by electron microscopy. Total RNA was isolated and microRNAs were analyzed by microarray analysis and real time RT-PCR. MicroRNA microarray analysis revealed that the microRNA profile of PC-3 released exosomes was similar to the profile of the corresponding parent cells. Nevertheless, a sorting of certain microRNAs into exosomes was observed, and low number microRNAs (microRNAs with a low number in their name) were found to be underrepresented in these vesicles. Moreover, the miRNA profile of PC-3 cells resembled the miRNA profile of RWPE-1 cells, though some miRNAs were found to be differently expressed in these cell lines. These results show that exosomes from PC-3 cells, in agreement with previous reports from other cell types, contain microRNAs. Furthermore, this study supports the idea that there is a sorting of microRNAs into exosomes and adds a new perspective by pointing at the underrepresentation of low number miRNAs in PC-3 released exosomes. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
156.9 (pelleting) / 276.6 (washing)
Protein markers
EV: CD63/ CD81/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
156.9
Wash: Rotor Type
SW40
Wash: adjusted k-factor
276.6
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9
Characterization: Particle analysis
EM
EM-type
transmission EM/ immune EM
Image type
Wide-field
EV120060 1/1 Homo sapiens Cell culture supernatant dUC Garnier D 2012 22%

Study summary

Full title
All authors
Garnier D, Magnus N, Lee TH, Bentley V, Meehan B, Milsom C, Montermini L, Kislinger T, Rak J
Journal
J Biol Chem
Abstract
Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant (show more...)Aggressive epithelial cancer cells frequently adopt mesenchymal characteristics and exhibit aberrant interactions with their surroundings, including the vasculature. Whether the release/uptake of extracellular vesicles (EVs) plays a role during these processes has not been studied. EVs are heterogeneous membrane structures that originate either at the surface (microparticles), or within (exosomes) activated or transformed cells, and are involved in intercellular trafficking of bioactive molecules. Here, we show that epithelial cancer cells (A431, DLD-1) adopt mesenchymal features (epithelial-to-mesenchymal transition-like state) upon activation of epidermal growth factor receptor (EGFR) coupled with blockade of E-cadherin. This treatment leads to a coordinated loss of EGFR and tissue factor (TF) from the plasma membrane and coincides with a surge in emission of small, exosome-like EVs containing both receptors. TF (but not EGFR) is selectively up-regulated in EVs produced by mesenchymal-like cancer cells and can be transferred to cultured endothelial cells rendering them highly procoagulant. We postulate that epithelial-to-mesenchymal transition-like changes may alter cancer cell interactions with the vascular systems through altered vesiculation and TF shedding. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes / extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: Flotilin1
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up
EV120119 2/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Ciravolo V 2012 22%

Study summary

Full title
All authors
Ciravolo V, Huber V, Ghedini GC, Venturelli E, Bianchi F, Campiglio M, Morelli D, Villa A, Della Mina P, Menard S, Filipazzi P, Rivoltini L, Tagliabue E, Pupa SM
Journal
J Cell Physiol
Abstract
Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and (show more...)Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
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
253.9 (pelleting) / 60.38 (washing)
Protein markers
EV: CD63/ Flotilin1/ AChE
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
253.9
Wash: Rotor Type
TLA100.3
Wash: adjusted k-factor
60.38
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ AChE
ELISA
Detected EV-associated proteins
AChE
Characterization: Particle analysis
EM
EM-type
immune EM
Image type
Close-up
EV120053 1/1 Homo sapiens Cell culture supernatant dUC Campanella C 2012 22%

Study summary

Full title
All authors
Campanella C, Bucchieri F, Merendino AM, Fucarino A, Burgio G, Corona DF, Barbieri G, David S, Farina F, Zummo G, de Macario EC, Macario AJ, Cappello F
Journal
PLoS One
Abstract
BACKGROUND: In a previous work we showed for the first time that human tumor cells secrete Hsp60 via (show more...)BACKGROUND: In a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here. PRINCIPAL FINDINGS: We found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle. CONCLUSIONS/SIGNIFICANCE: We propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
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
145.1 (pelleting)
Protein markers
EV: HSP60/ AChE
non-EV: None
Proteomics
no
Show all info
Study aim
Other/HSP60 trafficking
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
60Ti
Pelleting: adjusted k-factor
145.1
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
HSP60/ AChE
ELISA
Detected EV-associated proteins
HSP60/ AChE
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120052 1/2 Mus musculus Cell culture supernatant dUC Cai Z 2012 22%

Study summary

Full title
All authors
Cai Z, Yang F, Yu L, Yu Z, Jiang L, Wang Q, Yang Y, Wang L, Cao X, Wang J
Journal
J Immunol
Abstract
Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-ap (show more...)Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-?B pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD9/ HSP70/ TSG101/ Beta-actin/ CD8
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ HSP70/ TSG101/ Beta-actin/ CD8
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Beta-actin/ CD8
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120029 1/1 Mus musculus Cell culture supernatant dUC Bobrie A 2012 22%

Study summary

Full title
All authors
Bobrie A, Krumeich S, Reyal F, Recchi C, Moita LF, Seabra MC, Ostrowski M, Théry C
Journal
Cancer Res
Abstract
During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals (show more...)During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: Alix/ CD63/ TSG101/ "Hsc70 / MFGE8/ pro-MMP9"
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Alix/ CD63/ TSG101/ "Hsc70 / MFGE8/ pro-MMP9"
ELISA
Detected EV-associated proteins
Alix/ CD63/ TSG101/ "Hsc70 / MFGE8/ pro-MMP9"
Characterization: Particle analysis
EV120028 1/1 Mus musculus Cell culture supernatant 0.2 µm filter
dUC
Bellingham SA 2012 22%

Study summary

Full title
All authors
Bellingham SA, Coleman BM, Hill AF
Journal
Nucleic Acids Res
Abstract
Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The (show more...)Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The cellular prion protein, PrP(C), and the abnormal infectious form, PrP(Sc), are found associated with exosomes, which are small 50-130 nm vesicles released from cells. Exosomes also contain microRNAs (miRNAs), a class of non-coding RNA, and have been utilized to identify miRNA signatures for diagnosis of disease. While some miRNAs are deregulated in prion-infected brain tissue, the role of miRNA in circulating exosomes released during prion disease is unknown. Here, we investigated the miRNA profile in exosomes released from prion-infected neuronal cells. We performed the first small RNA deep sequencing study of exosomes and demonstrated that neuronal exosomes contain a diverse range of RNA species including retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, non-coding RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, silencing RNA as well as known and novel candidate miRNA. Significantly, we show that exosomes released by prion-infected neuronal cells have increased let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 levels with decreased miR-146 a levels compared to non-infected exosomes. Overall, these results demonstrate that circulating exosomes released during prion infection have a distinct miRNA signature that can be utilized for diagnosis and understanding pathogenic mechanisms in prion disease. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
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: Flotilin1/ TSG101
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120049 1/2 Homo sapiens Blood plasma 0.2 µm filter
Density cushion
dUC
Bastos-Amador P 2012 22%

Study summary

Full title
All authors
Bastos-Amador P, Royo F, Gonzalez E, Conde-Vancells J, Palomo-Diez L, Borras FE, Falcon-Perez JM
Journal
J Proteomics
Abstract
Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeu (show more...)Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeutic outcomes it is critical to control the quality of blood plasma. Clearly initiatives to improve the safety of blood transfusions will have a high economical and social impact. A detailed knowledge of the composition of healthy blood plasma is essential to facilitate such improvements. Apart from free proteins, lipids and metabolites, blood plasma also contains cell-derived microvesicles, including exosomes and microparticles from several different cellular origins. In this study, we have purified microvesicles smaller than 220nm from plasma of healthy donors and performed proteomic, ultra-structural, biochemical and functional analyses. We have detected 161 microvesicle-associated proteins, including many associated with the complement and coagulation signal-transduction cascades. Several proteases and protease inhibitors associated with acute phase responses were present, indicating that these microvesicles may be involved in these processes. There was a remarkably high variability in the protein content of plasma from different donors. In addition, we report that this variability could be relevant for their interaction with cellular systems. This work provides valuable information on plasma microvesicles and a foundation to understand microvesicle biology and clinical implications. (hide)
EV-METRIC
22% (60th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
DNF
Focus vesicles
microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + Density cushion + dUC
Protein markers
EV: CD63/ CD13
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD13
ELISA
Detected EV-associated proteins
CD13
Characterization: Particle analysis
EM
EM-type
cryo EM
Image type
Wide-field
EV120049 2/2 Homo sapiens Blood plasma 0.2 µm filter
dUC
Bastos-Amador P 2012 22%

Study summary

Full title
All authors
Bastos-Amador P, Royo F, Gonzalez E, Conde-Vancells J, Palomo-Diez L, Borras FE, Falcon-Perez JM
Journal
J Proteomics
Abstract
Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeu (show more...)Healthy blood plasma is required for several therapeutic procedures. To maximize successful therapeutic outcomes it is critical to control the quality of blood plasma. Clearly initiatives to improve the safety of blood transfusions will have a high economical and social impact. A detailed knowledge of the composition of healthy blood plasma is essential to facilitate such improvements. Apart from free proteins, lipids and metabolites, blood plasma also contains cell-derived microvesicles, including exosomes and microparticles from several different cellular origins. In this study, we have purified microvesicles smaller than 220nm from plasma of healthy donors and performed proteomic, ultra-structural, biochemical and functional analyses. We have detected 161 microvesicle-associated proteins, including many associated with the complement and coagulation signal-transduction cascades. Several proteases and protease inhibitors associated with acute phase responses were present, indicating that these microvesicles may be involved in these processes. There was a remarkably high variability in the protein content of plasma from different donors. In addition, we report that this variability could be relevant for their interaction with cellular systems. This work provides valuable information on plasma microvesicles and a foundation to understand microvesicle biology and clinical implications. (hide)
EV-METRIC
22% (60th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
DNF
Focus vesicles
microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Protein markers
EV: CD63/ CD81/ CD9/ Flotilin1/ TSG101/ CD13
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ Flotilin1/ TSG101/ CD13
ELISA
Detected EV-associated proteins
CD13
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EM
EM-type
cryo EM
Image type
Close-up
EV120027 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
Density cushion
dUC
Bastos-Amador P 2012 22%

Study summary

Full title
All authors
Bastos-Amador P, Pérez-Cabezas B, Izquierdo-Useros N, Puertas MC, Martinez-Picado J, Pujol-Borrell R, Naranjo-Gómez M, Borràs FE
Journal
J Leukoc Biol
Abstract
cDCs and pDCs differ in multiple aspects. Among those, antigen capture is a recognized feature of cD (show more...)cDCs and pDCs differ in multiple aspects. Among those, antigen capture is a recognized feature of cDCs, whereas pDCs display poor capacity to capture cell-derived antigens. However, animal models of organ transplantation suggested a role for pDCs in tolerance induction via phagocytosis of donor antigens. In a transplantation setting, microvesicles, such as apoptotic bodies and exosomes secreted by the graft, may be potential sources of alloantigen. Here, we tested the capacity of human pDCs to capture exosomes and apoptotic bodies from Jurkat T cells. Exosomes and apoptotic bodies were indeed captured by pDCs, although required longer times of incubation when compared with the highly endocytic cDCs. In cDCs and pDCs, exosome capture was more efficient than apoptotic bodies. Endocytosis inhibitors clearly impaired exosome capture by cDCs, although this could not be verified in pDCs as a result of cellular toxicity. Functionally, capture of Jurkat-derived exosomes did not induce nor prevent pDC maturation, and exosome-loaded pDCs induced T cell proliferation, suggesting a link between capture and presentation. Thus, exosomes and apoptotic bodies may be sources of antigen for human pDCs. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
exosomes / microvesicles / "Apoptotic bodies
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
Protein markers
EV: CD63/ CD81/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
75
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ Flotilin1
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
EV120026 1/1 Mus musculus Cell culture supernatant dUC Barraud-Lange V 2012 22%

Study summary

Full title
All authors
Barraud-Lange V, Chalas Boissonnas C, Serres C, Auer J, Schmitt A, Lefèvre B, Wolf JP, Ziyyat A
Journal
Reproduction
Abstract
Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicle (show more...)Spermatozoa undergo regulation of their functions along their lifespan through exchanges via vesicles or interactions with epithelial cells, in the epididymis, in the seminal fluid and in the female genital tract. Two different ways of oocyte membrane transfer to spermatozoa have been described: trogocytosis and exosomes. We here report an analysis of in vitro exchanges between the membranes of unfertilised oocytes and capacitated spermatozoa. We showed that optimum conditions are fulfilled when unfertilised oocytes interact with acrosome-reacted spermatozoa, a scenario mimicking the events occurring when the fertilising spermatozoon is inside the perivitelline space. Although CD9 tetraspanin is an essential molecule for fertilisation, exosome and trogocytosis transfer persists in Cd9-null oocytes in spite of their dramatic fusion failure. These exchanges are CD9 tetraspanin independent. We also confirm that mice sperm express CD9 tetraspanin and that when Cd9-null oocytes were inseminated with sperm covered with oocyte membrane materials, including CD9 tetraspanin, no rescue of the oocytes' fertilisability could be obtained. Thus, the existence of two ways of exchange between gametes during fertilisation suggests that these events could be of a physiological importance in this process. (hide)
EV-METRIC
22% (51st 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
Sample origin
DNF
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Other/Fertilization process
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV120025 1/3 Homo sapiens Semen dUC Aalberts M 2012 22%

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
22% (45th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Semen
Sample origin
DNF
Focus vesicles
Membrane(-derived) 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
dUC
Adj. k-factor
89.21 (pelleting)
Protein markers
EV: CD9/ PSCA
non-EV: None
Proteomics
no
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Semen
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
75
Pelleting: rotor type
TLA55
Pelleting: adjusted k-factor
89.21
Wash: Rotor Type
Fixed angle
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD9/ PSCA
ELISA
Characterization: Particle analysis
EV120169 1/1 Homo sapiens Cell culture supernatant Commercial Watson K 2012 17%

Study summary

Full title
All authors
Watson K, Koumangoye R, Thompson P, Sakwe AM, Patel T, Pratap S, Ochieng J
Journal
FEBS Lett
Abstract
Our goal in this study was to define the mechanisms by which fetuin-A mediates the adhesion of tumor (show more...)Our goal in this study was to define the mechanisms by which fetuin-A mediates the adhesion of tumor cells. The data show that in the absence of fetuin-A, detached tumor cells secrete exosomes that contain most of the known exosomal associated proteins but lack the capacity to mediate cellular adhesion. In the presence of fetuin-A, the cells secrete exosomes, which contain, in addition to the other exosomal proteins, fetuin-A, plasminogen and histones. These exosomes mediate adhesion and cell spreading. Plasminogen is a participant in this novel adhesion mechanism. The data suggest that these exosomes play a role in tumor progression. (hide)
EV-METRIC
17% (45th 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Commercial kit
ExoQuick
Characterization: Protein analysis
Characterization: Particle analysis
NTA
EV120090 2/3 Homo sapiens Cell culture supernatant 0.2 µm filter
Density cushion
Tolosa JM 2012 17%

Study summary

Full title
All authors
Tolosa JM, Schjenken JE, Clifton VL, Vargas A, Barbeau B, Lowry P, Maiti K, Smith R
Journal
Placenta
Abstract
OBJECTIVES: To examine whether syncytin-1 has immune regulatory functions and is carried by human pl (show more...)OBJECTIVES: To examine whether syncytin-1 has immune regulatory functions and is carried by human placental exosomes. Further, to examine whether corticotropin-releasing hormone (CRH) can induce the production of syncytin-1. STUDY DESIGN: Human placental exosomes were isolated from placental explant, primary trophoblast and BeWo cell cultures. The presence of exosomes was confirmed by transmission electron microscopy and western blotting. Exosomal protein was probed with 3 separate antibodies targeting syncytin-1. Syncytin-1 immunosuppression was tested, using either a syncytin-1 recombinant ectodomain protein or a synthetic peptide with the human syncytin-1 immunosuppressive domain sequence, in an in vitro human blood culture system immune challenged with LPS or PHA. The inhibition of cytokine production by syncytin-1 was determined by ELISA of TNF-?, IFN-? and CXCL10. BeWo cells were stimulated with CRH or vehicle for 24 h. mRNA and Protein was extracted from the cells for real-time PCR and western blotting analysis while exosomes were extracted from conditioned media for analysis by western blotting. RESULTS: Protein expression of syncytin-1 was detected in exosomes isolated from placental explants, primary trophoblast and BeWo cell cultures. Syncytin-1 recombinant ectodomain was also shown to inhibit the production of the Th1 cytokines TNF-? and IFN-? as well as the chemokine, CXCL10 in human blood cells. Finally, this study showed that syncytin-1 can be stimulated by CRH. CONCLUSIONS: The presence of syncytin-1 in placental exosomes provides a mechanism for syncytin-1 to reach and interact with target cells of the maternal immune system and represents a novel mechanism of endogenous retroviral mediated immunosuppression that may be relevant for maternal immune tolerance. (hide)
EV-METRIC
17% (45th 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
Sample origin
DNF
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
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
EV120087 2/3 Mus musculus Cell culture supernatant Commercial Singh PP 2012 17%

Study summary

Full title
All authors
Singh PP, Smith VL, Karakousis PC, Schorey JS
Journal
J Immunol
Abstract
More than 2 billion people are infected with Mycobacterium. tuberculosis; however, only 5-10% of tho (show more...)More than 2 billion people are infected with Mycobacterium. tuberculosis; however, only 5-10% of those infected will develop active disease. Recent data suggest that containment is controlled locally at the level of the granuloma and that granuloma architecture may differ even within a single infected individual. Formation of a granuloma likely requires exposure to mycobacterial components released from infected macrophages, but the mechanism of their release is still unclear. We hypothesize that exosomes, which are small membrane vesicles containing mycobacterial components released from infected macrophages, could promote cellular recruitment during granuloma formation. In support of this hypothesis, we found that C57BL/6 mouse-derived bone marrow macrophages treated with exosomes released from M. tuberculosis-infected RAW264.7 cells secrete significant levels of chemokines and can induce migration of CFSE-labeled macrophages and splenocytes. Exosomes isolated from the serum of M. bovis bacillus Calmette-Guérin-infected mice could also stimulate macrophage production of chemokines and cytokines ex vivo, but the level and type differed during the course of a 60-d infection. Of interest, the exosome concentration in serum correlated strongly with mouse bacterial load, suggesting some role in immune regulation. Finally, hollow fiber-based experiments indicated that macrophages treated with exosomes released from M. tuberculosis-infected cells could promote macrophage recruitment in vivo. Exosomes injected intranasally could also recruit CD11b(+) cells into the lung. Overall, our study suggests that exosomes may play an important role in recruiting and regulating host cells during an M. tuberculosis infection. (hide)
EV-METRIC
17% (45th 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Commercial kit
ExoQuick
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV120042 1/1 Rattus norvegicus/rattus Blood plasma Commercial Saludes JP 2012 17%

Study summary

Full title
All authors
Saludes JP, Morton LA, Ghosh N, Beninson LA, Chapman ER, Fleshner M, Yin H
Journal
ACS Chem Biol
Abstract
The generation of highly curved membranes is essential to cell growth, division, and movement. Recen (show more...)The generation of highly curved membranes is essential to cell growth, division, and movement. Recent research in the field is focused to answer questions related to the consequences of changes in the topology of the membrane once it is created, broadly termed as membrane curvature sensing. Most probes that are used to study curvature sensing are intact membrane active proteins such as DP1/Yop1p, ArfGAP1, BAR domains, and Synaptotagmin-I (Syt1). Taking a cue from nature, we created the cyclic peptide C2BL3C based on the membrane penetration C2B loop 3 of Syt1 via Click chemistry. Using a combination of spectroscopic techniques, we investigated the peptide-lipid interactions of this peptide with synthetic phospholipid vesicles and exosomes from rat blood plasma. We found that the macrocycle peptide probe was selective for lipid vesicles with highly curved surfaces (d < 100 nm). These results suggested that C2BL3C functions as a selective detector of highly curved phospholipid bilayers. (hide)
EV-METRIC
17% (52nd 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
Sample origin
DNF
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Commercial
Protein markers
EV: CD63/ Rab5b
non-EV: None
Proteomics
no
Show all info
Study aim
Other/Membrane curvature probe development
Sample
Species
Rattus norvegicus/rattus
Sample Type
Blood plasma
Isolation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Rab5b
ELISA
Detected EV-associated proteins
CD63/ Rab5b
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Wide-field
EV120137 1/2 Homo sapiens Cell culture supernatant 0.2 µm filter
0.45 µm
Density cushion
Immunoaffinity
Kadiu I 2012 17%

Study summary

Full title
All authors
Kadiu I, Narayanasamy P, Dash PK, Zhang W, Gendelman HE
Journal
J Immunol
Abstract
Exosomes and microvesicles (MV) are cell membranous sacs originating from multivesicular bodies and (show more...)Exosomes and microvesicles (MV) are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Their functional biology, however, remains incompletely understood. Macrophage exosomes and MV isolated by immunoaffinity and sucrose cushion centrifugation were characterized by morphologic, biochemical, and molecular assays. Lipidomic, proteomic, and cell biologic approaches uncovered novel processes by which exosomes and MV facilitate HIV-1 infection and dissemination. HIV-1 was entrapped in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV- and exosome-facilitated viral infections are affected by a range of cell surface receptors and adhesion proteins. HIV-1 containing exosomes readily completed its life cycle in human monocyte-derived macrophages but not in CD4(-) cells. The data support a significant role for exosomes as facilitators of viral infection. (hide)
EV-METRIC
17% (45th 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
Sample origin
DNF
Focus vesicles
exosomes / microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + 0.45 µm + Density cushion + Immunoaffinity
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Filtration steps
0.45µm > x > 0.22µm, 0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM/ scanning EM
Image type
Wide-field
EV120105 1/1 Cryptococcus neoformans
Bacillus anthracis
Bacteria 0.8 µm filter
dUC
Iodixanol-DG (valid.)
UF
Wolf JM 2012 14%

Study summary

Full title
All authors
Wolf JM, Rivera J, Casadevall A
Journal
Cell Microbiol
Abstract
For both pathogenic fungi and bacteria, extracellular vesicles have been shown to contain many micro (show more...)For both pathogenic fungi and bacteria, extracellular vesicles have been shown to contain many microbial components associated with virulence, suggesting a role in pathogenesis. However, there are many unresolved issues regarding vesicle synthesis and stability, including the fact that vesicular packaging for extracellular factors involved in virulence must also have a mechanism for vesicle unloading. Consequently, we studied the kinetics of vesicle production and stability using [1-(14) C] palmitic acid metabolic labelling and dynamic light scattering techniques. Cryptococcus neoformans vesicles were produced throughout all stages of fungal culture growth and they were stable once isolated. Density gradient analysis revealed that only a portion of the vesicle population carried cryptococcal polysaccharide, implying heterogeneity in vesicular cargo. Vesicle incubation with macrophages resulted in rapid vesicle instability, a phenomenon that was ultimately associated with serum albumin. Additionally, albumin, along with mouse serum and murine immunoglobulin destabilized Bacillus anthracis vesicles, but the effect was not observed with ovalbumin or keyhole limpet haemocyanin, demonstrating that this phenomenon is neither host-, microbe- nor protein-specific. Our findings strongly suggest that cryptococcal vesicles are short-lived in vivo and vesicle destabilization is mediated by albumin. The ability of albumin to promote vesicular offload through destabilization indicates a new activity for this abundant serum protein. (hide)
EV-METRIC
14% (56th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Bacteria
Sample origin
DNF
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
0.8 µm filter + dUC + Iodixanol-DG (valid.) + UF
Protein markers
EV: GXM
non-EV: None
Proteomics
no
Show all info
Study aim
Other/Bacterial vesicle stability
Sample
Species
Cryptococcus neoformans / Bacillus anthracis
Sample Type
Bacteria
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Density gradient
Only used for validation of main results
1
Density medium
Iodixanol
Lowest density fraction
10
Highest density fraction
35
Orientation
Bottom-up
Filtration steps
> 0.45 µm,
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
GXM
ELISA
Detected EV-associated proteins
GXM
Characterization: Particle analysis
DLS
EV120188 1/1 Paracoccidioides brasiliensis Fungus dUC
UF
Vallejo MC 2012 14%

Study summary

Full title
All authors
Vallejo MC, Nakayasu ES, Matsuo AL, Sobreira TJ, Longo LV, Ganiko L, Almeida IC, Puccia R
Journal
J Proteome Res
Abstract
Microorganisms release effector molecules that modulate the host machinery enabling survival, replic (show more...)Microorganisms release effector molecules that modulate the host machinery enabling survival, replication, and dissemination of a pathogen. Here we characterized the extracellular proteome of Paracoccidioides brasiliensis at its pathogenic yeast phase. Cell-free culture supernatants from the Pb18 isolate, cultivated in defined medium, were separated into vesicle and vesicle-free fractions, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. In vesicle and vesicle-free preparations we identified, respectively, 205 and 260 proteins with two or more peptides, including 120 overlapping identifications. Almost 70% of the sequences were predicted as secretory, mostly using nonconventional secretory pathways, and many have previously been localized to fungal cell walls. A total of 72 proteins were considered as commonly transported by extracellular vesicles, considering that orthologues have been reported in at least two other fungal species. These sequences were mostly related to translation, carbohydrate and protein metabolism, oxidation/reduction, transport, response to stress, and signaling. This unique proteomic analysis of extracellular vesicles and vesicle-free released proteins in a pathogenic fungus provides full comparison with other fungal extracellular vesicle proteomes and broadens the current view on fungal secretomes. (hide)
EV-METRIC
14% (64th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Fungus
Sample origin
DNF
Focus vesicles
Vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + UF
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Paracoccidioides brasiliensis
Sample Type
Fungus
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Characterization: Particle analysis
EV120101 1/1 Acholeplasma laidlawii PG8 Bacteria 0.2 µm filter
dUC
Chernov VM 2012 14%

Study summary

Full title
All authors
Chernov VM, Chernova OA, Mouzykantov AA, Baranova NB, Gorshkov OV, Trushin MV, Nesterova TN, Ponomareva AA
Journal
ScientificWorldJournal
Abstract
For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 ( (show more...)For the first time, the phytopathogenicity of extracellular vesicles of Acholeplasma laidlawii PG8 (a ubiquitous mycoplasma that is one of the five common species of cell culture contaminants and is a causative agent for phytomycoplasmoses) in Oryza sativa L. plants was studied. Data on the ability of extracellular vesicles of Acholeplasma laidlawii PG8 to penetrate from the nutrient medium into overground parts of Oryza sativa L. through the root system and to cause alterations in ultrastructural organization of the plants were presented. As a result of the analysis of ultrathin leaf sections of plants grown in medium with A. laidlawii PG8 vesicles, we detected significant changes in tissue ultrastructure characteristic to oxidative stress in plants as well as their cultivation along with bacterial cells. The presence of nucleotide sequences of some mycoplasma genes within extracellular vesicles of Acholeplasma laidlawii PG8 allowed a possibility to use PCR (with the following sequencing) to perform differential detection of cells and bacterial vesicles in samples under study. The obtained data may suggest the ability of extracellular vesicles of the mycoplasma to display in plants the features of infection from the viewpoint of virulence criteria--invasivity, infectivity--and toxigenicity--and to favor to bacterial phytopathogenicity. (hide)
EV-METRIC
14% (56th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Bacteria
Sample origin
DNF
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
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Acholeplasma laidlawii PG8
Sample Type
Bacteria
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
180
Filtration steps
0.22µm or 0.2µm
Characterization: Particle analysis
EM
EM-type
transmission EM/ atomic force EM
Image type
Close-up, Wide-field
EV120110 1/2 Rattus norvegicus/rattus Cell culture supernatant dUC
Sucrose-DG (valid.)
Zech D 2012 14%

Study summary

Full title
All authors
Zech D, Rana S, Büchler MW, Zöller M
Journal
Cell Commun Signal
Abstract
BACKGROUND: Tumor-exosomes being reported to suppress or promote a cancer-directed immune response, (show more...)BACKGROUND: Tumor-exosomes being reported to suppress or promote a cancer-directed immune response, we used exosomes of the rat pancreatic adenocarcinoma BSp73ASML (ASML) to evaluate, whether and which steps in immune response induction can be affected by tumor-exosomes and how the impaired responsiveness can be circumvented. RESULTS: ASML-exosomes bind to and are taken up by all leukocyte subpopulations in vivo and in vitro, uptake by CD11b+ leukocytes exceeding that by T and B cells. ASML-exosomes affect leukocyte proliferation via reduced CD44v6 up-regulation and lck, ZAP70 and ERK1,2 phosphorylation, which can be compensated by dendritic cells (DC). ASML-exosomes do not support Treg. Yet, impaired activation of anti-apoptotic signals is accompanied by slightly increased apoptosis susceptibility. IgM secretion is unaffected; NK and CTL activity are strengthened, ASML-exosomes co-operating with DC in CTL activation. ASML-exosomes transiently interfere with leukocyte migration by occupying migration-promoting receptors CD44, CD49d, CD62L and CD54 during binding/internalization. CONCLUSION: ASML-exosomes might well serve as adjuvant in immunotherapy as they support leukocyte effector functions and have only a minor impact on leukocyte activation, which can be overridden by DC. However, exosome-induced modulation of immune cells relies, at least in part, on exosome uptake and message transfer. This implies that depending on the individual tumor's exosome composition, exosomes may distinctly affect the immune system. Nonetheless, whether immunotherapy can profit from using tumor-exosomes as adjuvant can easily be settled beforehand in vitro. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Sucrose-DG (valid.)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
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
Density medium
Sucrose
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Particle analysis
EV120109 2/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Sucrose-DG (valid.)
Yoo CE 2012 14%

Study summary

Full title
All authors
Yoo CE, Kim G, Kim M, Park D, Kang HJ, Lee M, Huh N
Journal
Anal Biochem
Abstract
A direct extraction method was developed for exosomal microRNAs. After isolation of exosomes from hu (show more...)A direct extraction method was developed for exosomal microRNAs. After isolation of exosomes from human serum by immunoaffinity magnetic beads, microRNAs were extracted by just mixing beads with a lysis solution and heating without further purification. The lysis solution was composed of a nonionic detergent and salt (NaCl). The concentration of each component was optimized to maximize lysis efficiency and to inhibit adsorption of extracted microRNAs on beads. MicroRNAs extracted by this method could be quantitatively analyzed by qRT-PCR, indicating that the method could replace conventional methods for extracting microRNAs from immunobead-captured exosomes. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + Sucrose-DG (valid.)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
150
Density gradient
Only used for validation of main results
1
Density medium
Sucrose
Lowest density fraction
0.25
Highest density fraction
2
Orientation
Bottom-up
Filtration steps
0.22µm or 0.2µm
Characterization: Particle analysis
EV120171 1/1 Homo sapiens Saliva dUC Xiao H 2012 14%

Study summary

Full title
All authors
Xiao H, Wong DT
Journal
Anal Chim Acta
Abstract
Microvesicles (MVs) have been shown to affect the physiology of neighboring recipient cells in vario (show more...)Microvesicles (MVs) have been shown to affect the physiology of neighboring recipient cells in various ways. They play an important role in tumor progression/metastasis and angiogenesis in cancer and may be useful therapeutic tools, as well as a mechanism of cell-to-cell communication. They have been visioned as an important biomarker or biomarker source for the detection of different diseases. Human saliva is a biological fluid with enormous diagnostic potential, which harbors plenty of salivary MVs. The goal of this study is to investigate the proteomic profiling of MVs in human saliva through a simple preparation procedure by using filtration and centrifugation. Gel electrophoresis was combined with LC-MS/MS (liquid chromatography-mass spectrometry) for the proteomic analysis of MVs. After SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) protein separation, the whole lane was cut into 25 bands, and each band was subjected to in-gel trypsin digestion. The peptides extracted from each band were loaded to LC-MS/MS for protein identification. Through protein database search, 63 proteins were identified for human salivary MVs. Several members of different protein families were identified, including annexin, keratin, actin, immunoglobulin and S100. This study showed that although there was an overlap with the proteins from human saliva and salivary exosomes, salivary MVs contained their own unique proteins. These results will poise human salivary MVs as a non-invasive tool for the early detection of different diseases. (hide)
EV-METRIC
14% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Saliva
Sample origin
DNF
Focus vesicles
microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Saliva
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Characterization: Particle analysis
EV120168 1/1 Homo sapiens Urine dUC Wang Z 2012 14%

Study summary

Full title
All authors
Wang Z, Hill S, Luther JM, Hachey DL, Schey KL
Journal
Proteomics
Abstract
Exosomes are membrane vesicles that are secreted by cells upon fusion of multivesicular bodies with (show more...)Exosomes are membrane vesicles that are secreted by cells upon fusion of multivesicular bodies with the plasma membrane. Exosomal proteomics has emerged as a powerful approach to understand the molecular composition of exosomes and has potential to accelerate biomarker discovery. Different proteomic analysis methods have been previously employed to establish several exosome protein databases. In this study, TFE solution-phase digestion was compared with in-gel digestion and found to yield similar results. Proteomic analysis of urinary exosomes was performed by multidimensional protein identification technology (MudPIT) after TFE digestion. Nearly, 3280 proteins were identified from nine human urine samples with 31% overlap among nine samples. Gene ontology (GO) analysis, coupled with detection of all of the members of ESCRT machinery complex, supports the multivesicular origin of these particles. These results significantly expand the existing database of urinary exosome proteins. Our results also indicate that more than 1000 proteins can be detected from exosomes prepared from as little as 25 mL of urine. This study provides the largest set of proteins present in human urinary exosome proteomes, provides a valuable reference for future studies, and provides methods that can be applied to exosomal proteomic analysis from other tissue sources. (hide)
EV-METRIC
14% (41st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120166 1/1 Mus musculus Cell culture supernatant dUC Waldenström A 2012 14%

Study summary

Full title
All authors
Waldenström A, Gennebäck N, Hellman U, Ronquist G
Journal
PLoS One
Abstract
BACKGROUND: Shedding microvesicles are membrane released vesicles derived directly from the plasma m (show more...)BACKGROUND: Shedding microvesicles are membrane released vesicles derived directly from the plasma membrane. Exosomes are released membrane vesicles of late endosomal origin that share structural and biochemical characteristics with prostasomes. Microvesicles/exosomes can mediate messages between cells and affect various cell-related processes in their target cells. We describe newly detected microvesicles/exosomes from cardiomyocytes and depict some of their biological functions. METHODOLOGY/PRINCIPAL FINDINGS: Microvesicles/exosomes from media of cultured cardiomyocytes derived from adult mouse heart were isolated by differential centrifugation including preparative ultracentrifugation and identified by transmission electron microscopy and flow cytometry. They were surrounded by a bilayered membrane and flow cytometry revealed presence of both caveolin-3 and flotillin-1 while clathrin and annexin-2 were not detected. Microvesicle/exosome mRNA was identified and out of 1520 detected mRNA, 423 could be directly connected in a biological network. Furthermore, by a specific technique involving TDT polymerase, 343 different chromosomal DNA sequences were identified in the microvesicles/exosomes. Microvesicle/exosomal DNA transfer was possible into target fibroblasts, where exosomes stained for DNA were seen in the fibroblast cytosol and even in the nuclei. The gene expression was affected in fibroblasts transfected by microvesicles/exosomes and among 333 gene expression changes there were 175 upregulations and 158 downregulations compared with controls. CONCLUSIONS/SIGNIFICANCE: Our study suggests that microvesicles/exosomes released from cardiomyocytes, where we propose that exosomes derived from cardiomyocytes could be denoted cardiosomes, can be involved in a metabolic course of events in target cells by facilitating an array of metabolism-related processes including gene expression changes. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
Focus vesicles
microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.1 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
MLS50
Pelleting: adjusted k-factor
142.1
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Close-up
EV120094 1/2 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Wahlgren J 2012 14%

Study summary

Full title
All authors
Wahlgren J, De L Karlson T, Brisslert M, Vaziri Sani F, Telemo E, Sunnerhagen P, Valadi H
Journal
Nucleic Acids Res
Abstract
Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, se (show more...)Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC
Protein markers
EV: CD63/ CD81/ CD9
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Filtration steps
0.22µm or 0.2µm
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
DLS
Particle analysis: flow cytometry
EV120092 1/1 Homo sapiens Urine dUC van der Pol E 2012 14%

Study summary

Full title
All authors
van der Pol E, van Gemert MJ, Sturk A, Nieuwland R, van Leeuwen TG
Journal
J Thromb Haemost
Abstract
BACKGROUND: Microparticles and exosomes are cell-derived vesicles and potential biomarkers for disea (show more...)BACKGROUND: Microparticles and exosomes are cell-derived vesicles and potential biomarkers for disease. Recently, the Scientific Standardization Committee collaborative workshop of the ISTH initiated standardization of vesicle detection by flow cytometry with polystyrene beads. Because polystyrene beads have different optical properties from biological vesicles, and because the mechanisms causing the detection signal are incompletely understood, there are contradictions between expected and observed results. OBJECTIVES: To develop a model with which to relate the detection signal of a flow cytometer to the diameter of vesicles and clarify observed discrepancies. METHODS: We combined measurements of polystyrene and silica beads with an estimated refractive index of vesicles and performed Mie calculations of light scattering. RESULTS: We established the relationship between measured light scattering and the diameter of vesicles. The Megamix gating strategy proposed by the Scientific Standardization Committee selects single vesicles and cells with diameters between 800 and 2400 nm when applied on the forward-scattering detector of regular flow cytometers. Nevertheless, we demonstrated that, irrespective of the applied gating, multiple vesicles smaller than 220 nm or multiple 89-nm silica beads were counted as a single event signal at sufficiently high concentrations. CONCLUSIONS: Vesicle detection by flow cytometry is attributed to large single vesicles and swarm detection of smaller vesicles; that is, multiple vesicles are simultaneously illuminated by the laser beam and counted as a single event signal. Swarm detection allows the detection of smaller vesicles than previously thought possible, and explains the finding that flow cytometry underestimates the concentration of vesicles. (hide)
EV-METRIC
14% (41st 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
Sample origin
DNF
Focus vesicles
exosomes / microparticles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: None
non-EV: None
Proteomics
no
TEM measurements
55(mode)
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120164 1/1 Homo sapiens
Rattus norvegicus/rattus
Urine dUC van der Lubbe N 2012 14%

Study summary

Full title
All authors
van der Lubbe N, Jansen PM, Salih M, Fenton RA, van den Meiracker AH, Danser AH, Zietse R, Hoorn EJ
Journal
Hypertension
Abstract
Urinary exosomes are vesicles derived from renal tubular epithelial cells. Exosomes often contain se (show more...)Urinary exosomes are vesicles derived from renal tubular epithelial cells. Exosomes often contain several disease-associated proteins and are thus useful targets for identifying biomarkers of disease. Here, we hypothesized that the phosphorylated (active) form of the sodium chloride cotransporter (pNCC) or prostasin could serve as biomarkers for aldosteronism. We tested this in 2 animal models of aldosteronism (aldosterone infusion or low-sodium diet) and in patients with primary aldosteronism. Urinary exosomes were isolated from 24-hour urine or spot urine using ultracentrifugation. In rats, a normal or a high dose of aldosterone for 2, 3, or 8 days increased pNCC 3-fold in urinary exosomes (P<0.05 for all). A low-sodium diet also increased pNCC in urinary exosomes approximately 1.5-fold after 4 and after 8 days of treatment. The effects of these maneuvers on prostasin in urinary exosomes were less clear, showing a significant 1.5-fold increase only after 2 and 3 days of high-aldosterone infusion. In urinary exosomes of patients with primary aldosteronism, pNCC was 2.6-fold higher (P<0.05) while prostasin was 1.5-fold higher (P=0.07) than in patients with essential hypertension. Urinary exosomal pNCC and, to a lesser extent, prostasin are promising markers for aldosteronism in experimental animals and patients. These markers may be used to assess the biological activity of aldosterone and, potentially, as clinical biomarkers for primary aldosteronism. (hide)
EV-METRIC
14% (41st 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
Sample origin
DNF
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
104.8 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens / Rattus norvegicus/rattus
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
114
Pelleting: rotor type
45Ti
Pelleting: adjusted k-factor
104.8
Characterization: Particle analysis
EV120162 1/1 Homo sapiens Urine dUC Trnka P 2012 14%

Study summary

Full title
All authors
Trnka P, Ivanova L, Hiatt MJ, Matsell DG
Journal
Clin J Am Soc Nephrol
Abstract
BACKGROUND AND OBJECTIVES: Obstructive nephropathy is a leading cause of CKD in children. The assess (show more...)BACKGROUND AND OBJECTIVES: Obstructive nephropathy is a leading cause of CKD in children. The assessment of severity of renal impairment and the prediction of which children will progress to renal failure are, however, challenging. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This case-control study measured the urinary excretion of candidate biomarkers in 27 prevalent case-patients with posterior urethral valves (PUVs) and 20 age-matched controls, correlated their urinary concentration with GFR, and analyzed receiver-operating characteristic (ROC) curve and regression analyses to assess their performance as tests for low GFR. RESULTS: The median urinary protein-to-creatinine ratio was higher in children with PUV (45 g/mol; range, 5-361 g/mol) than in controls (7 g/mol; range, 3-43 g/mol) (P<0.01) and correlated inversely with renal function (r = -0.44; P<0.05). In whole urine, excretion of aquaporin-2 was significantly decreased, whereas that of TGF? and L1 cell adhesion molecule (L1CAM) was significantly increased. Whole-urine TGF? excretion correlated inversely with GFR (r = -0.53; P<0.05). As tests for low GFR, whole-urine TGF?, L1CAM, and urinary protein-to-creatinine ratio performed best, with areas under the ROC curves of 0.788, 0.795, and 0.814, respectively. By linear regression analysis, whole-urine TGF?, L1CAM, and urinary protein-to-creatinine ratio were associated with low GFR in the case-patients. CONCLUSIONS: Candidate biomarkers of obstructive nephropathy can be readily measured in whole urine and in urine exosomes. In boys with PUV, these biomarkers correlate with GFR. (hide)
EV-METRIC
14% (41st 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
Sample origin
DNF
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
138.3 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
SW40
Pelleting: adjusted k-factor
138.3
Characterization: Particle analysis
EV120152 1/1 Homo sapiens Urine dUC Pisitkun T 2012 14%

Study summary

Full title
All authors
Pisitkun T, Gandolfo MT, Das S, Knepper MA, Bagnasco SM
Journal
Proteomics Clin Appl
Abstract
PURPOSE: In mass spectrometry (MS)-based studies to discover urinary protein biomarkers, an importan (show more...)PURPOSE: In mass spectrometry (MS)-based studies to discover urinary protein biomarkers, an important question is how to analyze the data to find the most promising potential biomarkers to be advanced to large-scale validation studies. Here, we describe a systems biology-based approach to address this question. EXPERIMENTAL DESIGN: We analyzed large-scale liquid chromatography-tandem mass spectrometry (LC-MS/MS) data of urinary exosomes from renal allograft recipients with biopsy-proven evidence of immunological rejection or tubular injury (TI). We asked whether bioinformatic analysis of urinary exosomal proteins can identify biological-process based protein groups that correlate with biopsy findings and whether the protein groups fit with general knowledge of the pathophysiological mechanisms involved. RESULTS: LC-MS/MS analysis of urinary exosomal proteomes identified more than 1000 proteins in each pathologic group. These protein lists were analyzed computationally to identify the Biological Process and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway terms that are significantly associated with each pathological group. Among the most informative terms for each group were: sodium ion transport for TI; immune response for all rejection; epithelial cell differentiation for cell-mediated rejection; and acute inflammatory response for antibody-mediated rejection. Based on these terms, candidate biomarkers were identified using a novel strategy to allow a dichotomous classification between different pathologic categories. CONCLUSIONS AND CLINICAL RELEVANCE: The terms and candidate biomarkers identified make rational connections to pathophysiological mechanisms, suggesting that the described bioinformatic approach will be useful in advancing large-scale biomarker identification studies toward a validation phase. (hide)
EV-METRIC
14% (41st 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
Sample origin
DNF
Focus vesicles
exosomes
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Homo sapiens
Sample Type
Urine
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Characterization: Particle analysis
EV120151 1/2 Rattus norvegicus/rattus Cell culture supernatant dUC Palmisano G 2012 14%

Study summary

Full title
All authors
Palmisano G, Jensen SS, Le Bihan MC, Lainé J, McGuire JN, Pociot F, Larsen MR
Journal
Mol Cell Proteomics
Abstract
Microparticles and exosomes are two of the most well characterized membrane-derived microvesicles re (show more...)Microparticles and exosomes are two of the most well characterized membrane-derived microvesicles released either directly from the plasma membrane or released through the fusion of intracellular multivesicular bodies with the plasma membrane, respectively. They are thought to be involved in many significant biological processes such as cell to cell communication, rescue from apoptosis, and immunological responses. Here we report for the first time a quantitative study of proteins from ?-cell-derived microvesicles generated after cytokine induced apoptosis using stable isotope labeled amino acids in cell culture combined with mass spectrometry. We identified and quantified a large number of ?-cell-specific proteins and proteins previously described in microvesicles from other cell types in addition to new proteins located to these vesicles. In addition, we quantified specific sites of protein phosphorylation and N-linked sialylation in proteins associated with microvesicles from ?-cells. Using pathway analysis software, we were able to map the most distinctive changes between microvesicles generated during growth and after cytokine stimulation to several cell death and cell signaling molecules including tumor necrosis factor receptor superfamily member 1A, tumor necrosis factor, ?-induced protein 3, tumor necrosis factor-interacting kinase receptor-interacting serine-threonine kinase 1, and intercellular adhesion molecule 1. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
Focus vesicles
microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Omics
Sample
Species
Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
60
Characterization: Protein analysis
Characterization: Particle analysis
EV120080 1/1 Homo sapiens
Rattus norvegicus/rattus
Cell culture supernatant dUC
Sucrose-DG
Ngora H 2012 14%

Study summary

Full title
All authors
Ngora H, Galli UM, Miyazaki K, Zöller M
Journal
Neoplasia
Abstract
Metastasis-associated C4.4A, which becomes upregulated during wound healing and, in some tumors, dur (show more...)Metastasis-associated C4.4A, which becomes upregulated during wound healing and, in some tumors, during tumor progression, is known to be frequently associated with hypoxia. With the function of C4.4A still unknown, we explored the impact of hypoxia on C4.4A expression and functional activity. Metastatic rat and human tumor lines upregulate C4.4A expression when cultured in the presence of CoCl(2). Although hypoxia-inducible factor 1? (HIF-1?) becomes upregulated concomitantly, HIF-1? did not induce C4.4A transcription. Instead, hypoxia-induced C4.4A up-regulation promoted in vivo and in vitro wound healing, where increased migration on the C4.4A ligands laminin-111 and -332 was observed after a transient period of pronounced binding. Increased migration was accompanied by C4.4A associating with ?(6)?(4), MT1-MMP1, and TACE and by laminin fragmentation. Hypoxia also promoted the release of C4.4A in exosomes and TACE-mediated C4.4A shedding. The association of C4.4A with ?(6)?(4) and MT1-MMP1 was maintained in exosomes and exosomal ?(6)?(4)- and MT1-MMP1-associated C4.4A but not shed C4.4A sufficient for laminin degradation. Hypoxia-induced recruitment of ?(6)?(4) toward raft-located C4.4A, MT1-MMP, and TACE allows for a shift from adhesion to motility, which is supported by laminin degradation. These findings provide the first explanation for the C4.4A contribution to wound healing and metastasis. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
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: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens / Rattus norvegicus/rattus
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
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
Density medium
Sucrose
Lowest density fraction
0.25
Highest density fraction
2.5
Orientation
Bottom-up
Characterization: Particle analysis
EV120149 1/1 Mus musculus Cell culture supernatant dUC Nanjundappa RH 2012 14%

Study summary

Full title
All authors
Nanjundappa RH, Wang R, Xie Y, Umeshappa CS, Xiang J
Journal
Vaccine
Abstract
The limitations of highly active anti-retroviral therapy have necessitated the development of altern (show more...)The limitations of highly active anti-retroviral therapy have necessitated the development of alternative therapeutics for human immunodeficiency virus type-1 (HIV-1)-infected patients with dysfunctional dendritic cells (DCs) and CD4(+) T cell deficiency. We previously demonstrated that HIV-1 Gp120-specific T cell-based Gp120-Texo vaccine by using ConA-stimulated C57BL/6 (B6) mouse CD8(+) T (ConA-T) cells with uptake of pcDNA(Gp120)-transfected B6 mouse DC line DC2.4 (DC2.4(Gp120))-released exosomes (EXO(Gp120)) was capable of stimulating DC and CD4(+) T cell-independent CD8(+) cytotoxic T lymphocyte (CTL) responses detected in wild-type B6 mice using non-specific PE-anti-CD44 and anti-IFN-? antibody staining by flow cytometry. To assess effectiveness of Gp120-Texo vaccine in transgenic (Tg) HLA-A2 mice mimicking the human situation, we constructed adenoviral vector AdV(Gp120) expressing HIV-1 GP120 by recombinant DNA technology, and generated Gp120-Texo vaccine by using Tg HLA-A2 mouse CD8(+) ConA-T cells with uptake of AdV(Gp120)-transfected HLA-A2 mouse bone marrow DC (DC(Gp120))-released EXO(Gp120). We then performed animal studies to assess Gp120-Texo-induced stimulation of Gp120-specific CTL responses and antitumor immunity in Tg HLA-A2 mice. We demonstrate that Gp120-Texo vaccine stimulates Gp120-specific CTL responses detected in Tg HLA-A2 mice using Gp120-specific PE-HLA-A2/Gp120 peptide (KLTPLCVTL) tetramer staining by flow cytometry. These Gp120-specific CTLs are capable of further differentiating into functional effectors with killing activity to Gp120 peptide-pulsed splenocytes in vivo. In addition, Gp120-Texo vaccine also induces Gp120-specific preventive, therapeutic (for 6 day tumor lung metastasis) and CD4(+) T cell-independent long-term immunity against B16 melanoma BL6-10(Gp120/A2Kb) expressing both Gp120 and A2Kb (?1 and ?2 domains of HLA-A2 and ?3 domain of H-2K(b)) in Tg HLA-A2 mice. Taken together, the novel CD8(+) Gp120-Texo vaccine capable of stimulating efficient CD4(+) T cell-independent Gp120-specific CD8(+) CTL responses leading to therapeutic and long-term immunity in Tg HLA-A2 mice may represent a new immunotherapeutic vaccine for treatment of HIV-1 patients with CD4(+) T cell deficiency. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
Focus vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Protein markers
EV: GP120/ MHC1/ Iab/ CD11c/ CD54/ CD80
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Western Blot
Detected EV-associated proteins
GP120/ MHC1/ Iab/ CD11c/ CD54/ CD80
ELISA
Detected EV-associated proteins
GP120/ MHC1/ Iab/ CD11c/ CD54/ CD80
Flow cytometry specific beads
Selected surface protein(s)
Yes
Characterization: Particle analysis
Particle analysis: flow cytometry
EV120106 1/4 Homo sapiens Cell culture supernatant dUC Maguire CA 2012 14%

Study summary

Full title
All authors
Maguire CA, Balaj L, Sivaraman S, Crommentuijn MH, Ericsson M, Mincheva-Nilsson L, Baranov V, Gianni D, Tannous BA, Sena-Esteves M, Breakefield XO, Skog J
Journal
Mol Ther
Abstract
Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured (show more...)Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
Focus vesicles
microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
142.6 (pelleting) / 104.9 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
80
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
142.6
Wash: volume per pellet (ml)
12
Wash: Rotor Type
MLA55
Wash: adjusted k-factor
104.9
Characterization: Particle analysis
EV120106 4/4 Homo sapiens Cell culture supernatant dUC
Iodixanol-DG (valid.)
Maguire CA 2012 14%

Study summary

Full title
All authors
Maguire CA, Balaj L, Sivaraman S, Crommentuijn MH, Ericsson M, Mincheva-Nilsson L, Baranov V, Gianni D, Tannous BA, Sena-Esteves M, Breakefield XO, Skog J
Journal
Mol Ther
Abstract
Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured (show more...)Adeno-associated virus (AAV) vectors have shown remarkable efficiency for gene delivery to cultured cells and in animal models of human disease. However, limitations to AAV vectored gene transfer exist after intravenous transfer, including off-target gene delivery (e.g., liver) and low transduction of target tissue. Here, we show that during production, a fraction of AAV vectors are associated with microvesicles/exosomes, termed vexosomes (vector-exosomes). AAV capsids associated with the surface and in the interior of microvesicles were visualized using electron microscopy. In cultured cells, vexosomes outperformed conventionally purified AAV vectors in transduction efficiency. We found that purified vexosomes were more resistant to a neutralizing anti-AAV antibody compared to conventionally purified AAV. Finally, we show that vexosomes bound to magnetic beads can be attracted to a magnetized area in cultured cells. Vexosomes represent a unique entity which offers a promising strategy to improve gene delivery. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
Focus vesicles
microvesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC + Iodixanol-DG (valid.)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Pelleting: time(min)
25
Density gradient
Only used for validation of main results
1
Density medium
Iodixanol
Lowest density fraction
6
Highest density fraction
18
Orientation
Top-down
Characterization: Particle analysis
NTA
EV120143 1/1 Klebsiella pneumoniae Bacteria 0.2 µm filter
dUC
UF
Lee JC 2012 14%

Study summary

Full title
All authors
Lee JC, Lee EJ, Lee JH, Jun SH, Choi CW, Kim SI, Kang SS, Hyun S
Journal
FEMS Microbiol Lett
Abstract
Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehic (show more...)Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1? and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response. (hide)
EV-METRIC
14% (56th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Bacteria
Sample origin
DNF
Focus vesicles
OMV
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
0.2 µm filter + dUC + UF
Protein markers
EV: None
non-EV: None
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Klebsiella pneumoniae
Sample Type
Bacteria
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
180
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120123 1/1 Gallus gallus Cell culture supernatant 0.2 µm filter
dUC
del Cacho E 2012 14%

Study summary

Full title
All authors
del Cacho E, Gallego M, Lee SH, Lillehoj HS, Quilez J, Lillehoj EP, Sánchez-Acedo C
Journal
Infect Immun
Abstract
This study describes a novel immunization strategy against avian coccidiosis using exosomes derived (show more...)This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting T(h)1 cytokines, T(h)2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the T(h)1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the T(h)2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible. (hide)
EV-METRIC
14% (40th 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
Sample origin
DNF
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
122.2 (pelleting)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Gallus gallus
Sample Type
Cell culture supernatant
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
60
Pelleting: rotor type
70.1Ti
Pelleting: adjusted k-factor
122.2
Filtration steps
0.22µm or 0.2µm
Characterization: Particle analysis
EV120112 1/4 Equus caballus Serum dUC da Silveira JC 2012 14%

Study summary

Full title
All authors
da Silveira JC, Veeramachaneni DN, Winger QA, Carnevale EM, Bouma GJ
Journal
Biol Reprod
Abstract
Proper cell communication within the ovarian follicle is critical for the growth and maturation of a (show more...)Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid. (hide)
EV-METRIC
14% (63rd 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
Sample origin
DNF
Focus vesicles
Vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
138.6 (pelleting) / 138.6 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Omics
Sample
Species
Equus caballus
Sample Type
Serum
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW55
Pelleting: adjusted k-factor
138.6
Wash: Rotor Type
SW55
Wash: adjusted k-factor
138.6
Characterization: Particle analysis
EV120112 2/4 Equus caballus Follicular fluid dUC da Silveira JC 2012 14%

Study summary

Full title
All authors
da Silveira JC, Veeramachaneni DN, Winger QA, Carnevale EM, Bouma GJ
Journal
Biol Reprod
Abstract
Proper cell communication within the ovarian follicle is critical for the growth and maturation of a (show more...)Proper cell communication within the ovarian follicle is critical for the growth and maturation of a healthy oocyte that can be fertilized and develop into an embryo. Cell communication within the follicle involves many signaling molecules and is affected by maternal age. Recent studies indicate that cell communication can be mediated through secretion and uptake of small membrane-enclosed vesicles. The goals of this study were to 1) identify cell-secreted vesicles (microvesicles and exosomes) containing miRNAs and proteins within ovarian follicular fluid and 2) determine if miRNA level differs in exosomes isolated from follicular fluid in young compared to old mares. We demonstrate the presence of vesicles resembling microvesicles and exosomes in ovarian follicular fluid using transmission electron microscopy and CD63-positive and RNA containing vesicles using flow cytometry. Moreover, proteomics analysis reveals that follicular fluid-isolated exosomes contain both known exosomal proteins and proteins not previously reported in isolated exosomes. MicroRNAs were detected in microvesicle and exosomes preparations isolated from follicular fluid by real-time PCR analysis. Uptake of fluorescent-labeled microvesicles by granulosa cells was examined using in vitro and in vivo approaches. MicroRNA expression profiling reveals that miRNAs in microvesicle and exosome preparations isolated from follicular fluid also are present within surrounding granulosa and cumulus cells. These studies revealed that cell communication within the mammalian ovarian follicle may involve transfer of bioactive material by microvesicles and exosomes. Finally, miRNAs present in exosomes from ovarian follicular fluid varied with the age of the mare, and a number of different miRNAs were detected in young vs. old mare follicular fluid. (hide)
EV-METRIC
14% (25th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Follicular fluid
Sample origin
DNF
Focus vesicles
Vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
138.6 (pelleting) / 138.6 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Omics
Sample
Species
Equus caballus
Sample Type
Follicular fluid
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW55
Pelleting: adjusted k-factor
138.6
Wash: Rotor Type
SW55
Wash: adjusted k-factor
138.6
Characterization: Particle analysis
EM
EM-type
transmission EM
Image type
Wide-field
EV120119 1/2 Homo sapiens Serum dUC Ciravolo V 2012 14%

Study summary

Full title
All authors
Ciravolo V, Huber V, Ghedini GC, Venturelli E, Bianchi F, Campiglio M, Morelli D, Villa A, Della Mina P, Menard S, Filipazzi P, Rivoltini L, Tagliabue E, Pupa SM
Journal
J Cell Physiol
Abstract
Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and (show more...)Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness. (hide)
EV-METRIC
14% (63rd 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
Sample origin
DNF
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
253.9 (pelleting) / 60.38 (washing)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Serum
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW28
Pelleting: adjusted k-factor
253.9
Wash: Rotor Type
TLA100.3
Wash: adjusted k-factor
60.38