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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140233	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Xiao W	2014	22%
Study summary Full title Effects of the epigenetic drug MS-275 on the release and function of exosome-related immune molecules in hepatocellular carcinoma cells All authors Xiao W, Dong W, Zhang C, Saren G, Geng P, Zhao H, Li Q, Zhu J, Li G, Zhang S, Ye M Journal Eur J Med Res Abstract BACKGROUND: Tumor-derived exosomes have been viewed as a source of tumor antigens that can be used t (show more...)BACKGROUND: Tumor-derived exosomes have been viewed as a source of tumor antigens that can be used to induce anti-tumor immune responses. In the current study, we aim to investigate the regulatory effect of the epigenetic drug MS-275 on hepatoma G2 (HepG2) cell-derived exosomes, especially for their immunostimulatory properties and alteration of some non-specific immune protein expression, such as heat shock protein (HSP) 70, major histocompatibility complex (MHC) class I polypeptide-related sequence A (MICA) and MICB. METHODS: MS-275 was used to modulate the secretion of exosomes in human HepG2 cells, and exosomes from untreated HepG2 cells served as negative controls. RT-PCR was used to test the expression of HSP70, MICA and MICB in HepG2 cells. Immunogold labeling of exosomes and western blotting analysis were carried out to compare the expression of HSP70, MICA and MICB proteins in exosomes with or without MS-275 treatment. A natural killer (NK) cell cytotoxicity assay and peripheral blood mononuclear cell (PBMC) proliferation assay were used to evaluate the effect of MS-275 on the immunostimulatory ability of exosomes. RESULTS: Immunogold labeling and western blot analysis showed that modification of MS-275 increased the expression of HSP70 and MICB in exosomes. RT-PCR showed the mRNA levels of HSP70 and MICB were upregulated in HepG2 cells and were consistent with their protein levels in exosomes. The exosomes modified by MS-275 could significantly increase the cytotoxicity of NK cells and proliferation of PBMC (P < 0.05). CONCLUSIONS: The non-specific immune response of exosomes derived from HepG2 cells could be enhanced with treatment by the histone deacetylase inhibitor (HDACi) drug MS-275; this could provide a potential tumor vaccine strategy against liver cancer. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: HSP70/ Beta-actin non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ Beta-actin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein HSP70 Image type Wide-field

	EV140225	1/1	Homo sapiens Mus musculus	NAY	(d)(U)C	Tsunemi T	2014	22%
Study summary Full title ATP13A2/PARK9 Regulates Secretion of Exosomes and alpha-Synuclein All authors Tsunemi T, Hamada K, Krainc D Journal J Neurosci Abstract Kufor-Rakeb syndrome (KRS) is caused by loss-of-function mutations in ATP13A2 (PARK9) and characteri (show more...)Kufor-Rakeb syndrome (KRS) is caused by loss-of-function mutations in ATP13A2 (PARK9) and characterized by juvenile-onset parkinsonism, pyramidal signs, and cognitive decline. Previous studies suggested that PARK9 deficiency causes lysosomal dysfunction and ?-synuclein (?-syn) accumulation, whereas PARK9 overexpression suppresses toxicity of ?-syn. However, the precise mechanism of PARK9 effect on lysosomes and ?-syn has been unknown. Here, we found that overexpressed PARK9 localized to multivesicular bodies (MVBs) in the human H4 cell line. The results from patient fibroblasts showed that loss of PARK9 function leads to decreased number of the intraluminal vesicles in MVBs and diminished release of exosomes into culture media. By contrast, overexpression of PARK9 results in increased release of exosomes in H4 cells and mouse primary cortical neurons. Moreover, loss of PARK9 function resulted in decreased secretion of ?-syn into extracellular space, whereas overexpressed PARK9 promotes secretion of ?-syn, at least in part via exosomes. Finally, we found that PARK9 regulates exosome biogenesis through functional interaction with the endosomal sorting complex required for transport machinery. Together, these data suggest the involvement of PARK9 in the biogenesis of exosomes and ?-syn secretion and raise a possibility that disruption of these pathways in patients with KRS contributes to the disease pathogenesis. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 59.72 (pelleting) / 59.72 (washing) Protein markers EV: Alix/ TSG101/ Flotilin1/ CD63/ Alpha-synuclein non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Pelleting: rotor type TLA110 Pelleting: adjusted k-factor 59.72 Wash: Rotor Type TLA110 Wash: adjusted k-factor 59.72 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ Flotilin1/ TSG101/ Alpha-synuclein ELISA Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ Flotilin1/ TSG101/ Alpha-synuclein Characterization: Particle analysis NTA

	EV140126	1/2	Homo sapiens	NAY	(d)(U)C Filtration	Soldevilla B	2014	22%
Study summary Full title Tumor-derived exosomes are enriched in DeltaNp73, which promotes oncogenic potential in acceptor cells and correlates with patient survival All authors Soldevilla B, Rodríguez M, San Millán C, García V, Fernández-Periañez R, Gil-Calderón B, Martín P, García-Grande A, Silva J, Bonilla F, Domínguez G Journal Hum Mol Genet Abstract Tumor-derived exosomes are emerging as local and systemic cell-to-cell mediators of oncogenic inform (show more...)Tumor-derived exosomes are emerging as local and systemic cell-to-cell mediators of oncogenic information through the horizontal transfer of mRNAs, microRNAs and proteins during tumorigenesis. The exosomal content has been described as biologically active when taken up by the recipient cell. Identifying the specific molecular cargo of exosomes will help to determine their function in specific steps of the tumorigenic process. Here we evaluate whether ?Np73 is selectively packaged in tumor-derived exosomes, its function in the acceptor cells in vitro and in vivo and its prognosis potential in cancer. ?Np73 messenger is enriched in tumor-derived exosomes, suggesting its active sorting in these microvesicles. We observed the transmission of this exosome cargo to different cell types and how it confers proliferation potential and chemoresistance to the acceptor cells in vitro and in animal models. Finally, our data support the potential prognostic value of exosomal ?Np73 in colon cancer patients. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD63/ CD9 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD9 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140097	1/1	Homo sapiens	NAY	(d)(U)C	Shimbo K	2014	22%
Study summary Full title Exosome-formed synthetic microRNA-143 is transferred to osteosarcoma cells and inhibits their migration All authors Shimbo K, Miyaki S, Ishitobi H, Kato Y, Kubo T, Shimose S, Ochi M Journal Biochem Biophys Res Commun Abstract MicroRNAs (miRNAs) have emerged as potential anticancer agents, but their clinical application is li (show more...)MicroRNAs (miRNAs) have emerged as potential anticancer agents, but their clinical application is limited by the lack of an effective delivery system to tumors. Exosomes are small vesicles that play important roles in intercellular communication. Here, we show that synthetic miR-143 introduced into cells is released enveloped in exosomes and that the secreted exosome-formed miR-143 is transferred to osteosarcoma cells. The delivery of exosome-formed miR-143 significantly reduced the migration of osteosarcoma cells. The delivery efficiency of exosome-formed miR-143 was less than that achieved with lipofection, but the migratory potential of osteosarcoma cells was similarly inhibited after both strategies. Our results suggest that exosomes can deliver synthetic miR-143 and are a potentially efficient and functional delivery system. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD81/ Flotilin1 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD81/ Flotilin1 Characterization: Particle analysis TRPS

	EV140095	1/1	Homo sapiens	NAY	(d)(U)C	Sharghi-Namini S	2014	22%
Study summary Full title Dll4-containing exosomes induce capillary sprout retraction in a 3D microenvironment All authors Sharghi-Namini S, Tan E, Ong LL, Ge R, Asada HH Journal Sci Rep Abstract Delta-like 4 (Dll4), a membrane-bound Notch ligand, plays a fundamental role in vascular development (show more...)Delta-like 4 (Dll4), a membrane-bound Notch ligand, plays a fundamental role in vascular development and angiogenesis. Dll4 is highly expressed in capillary endothelial tip cells and is involved in suppressing neighboring stalk cells to become tip cells during angiogenesis. Dll4-Notch signaling is mediated either by direct cell-cell contact or by Dll4-containing exosomes from a distance. However, whether Dll4-containing exosomes influence tip cells of existing capillaries is unknown. Using a 3D microfluidic device and time-lapse confocal microscopy, we show here for the first time that Dll4-containing exosomes causes tip cells to lose their filopodia and trigger capillary sprout retraction in collagen matrix. We demonstrate that Dll4 exosomes can freely travel through 3D collagen matrix and transfer Dll4 protein to distant tip cells. Upon reaching endothelial sprout, it causes filopodia and tip cell retraction. Continuous application of Dll4 exosomes from a distance lead to significant reduction of sprout formation. This effect correlates with Notch signaling activation upon Dll4-containing exosome interaction with recipient endothelial cells. Furthermore, we show that Dll4-containing exosomes increase endothelial cell motility while suppressing their proliferation. These data revealed novel functions of Dll4 in angiogenesis through exosomes. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ ICAM1/ GAPDH/ Alix/ LAMP1/ Beta-actin non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ TSG101/ Beta-actin/ ICAM1/ GAPDH/ LAMP1 ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin/ ICAM1/ GAPDH/ LAMP1 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV140217	1/5	Homo sapiens	NAY	(d)(U)C Filtration UF	Sedlik C	2014	22%
Study summary Full title Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms All authors Sedlik C, Vigneron J, Torrieri-Dramard L, Pitoiset F, Denizeau J, Chesneau C, de la Rochere P, Lantz O, Thery C, Bellier B Journal J Extracell Vesicles Abstract The induction of an active immune response to control or eliminate tumours is still an unfulfilled c (show more...)The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8(+) T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4(+) T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 14899 (pelleting) Protein markers EV: OVA non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type SW32.1 Pelleting: adjusted k-factor 14899 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins OVA ELISA Antibody details provided? No Detected EV-associated proteins OVA Characterization: Particle analysis NTA

	EV140217	2/5	Homo sapiens	NAY	(d)(U)C Filtration UF	Sedlik C	2014	22%
Study summary Full title Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms All authors Sedlik C, Vigneron J, Torrieri-Dramard L, Pitoiset F, Denizeau J, Chesneau C, de la Rochere P, Lantz O, Thery C, Bellier B Journal J Extracell Vesicles Abstract The induction of an active immune response to control or eliminate tumours is still an unfulfilled c (show more...)The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8(+) T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4(+) T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 2979 (pelleting) Protein markers EV: OVA non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 40 Pelleting: rotor type SW32.1 Pelleting: adjusted k-factor 2979. Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins OVA ELISA Antibody details provided? No Detected EV-associated proteins OVA Characterization: Particle analysis NTA

	EV140217	3/5	Homo sapiens	NAY	(d)(U)C Filtration UF	Sedlik C	2014	22%
Study summary Full title Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms All authors Sedlik C, Vigneron J, Torrieri-Dramard L, Pitoiset F, Denizeau J, Chesneau C, de la Rochere P, Lantz O, Thery C, Bellier B Journal J Extracell Vesicles Abstract The induction of an active immune response to control or eliminate tumours is still an unfulfilled c (show more...)The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8(+) T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4(+) T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 298 (pelleting) Protein markers EV: OVA non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 75 Pelleting: rotor type SW32.1 Pelleting: adjusted k-factor 298 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins OVA ELISA Antibody details provided? No Detected EV-associated proteins OVA Characterization: Particle analysis NTA

	EV140217	4/5	Homo sapiens	NAY	(d)(U)C Filtration UF	Sedlik C	2014	22%
Study summary Full title Different immunogenicity but similar antitumor efficacy of two DNA vaccines coding for an antigen secreted in different membrane vesicle-associated forms All authors Sedlik C, Vigneron J, Torrieri-Dramard L, Pitoiset F, Denizeau J, Chesneau C, de la Rochere P, Lantz O, Thery C, Bellier B Journal J Extracell Vesicles Abstract The induction of an active immune response to control or eliminate tumours is still an unfulfilled c (show more...)The induction of an active immune response to control or eliminate tumours is still an unfulfilled challenge. We focused on plasmid DNA vaccines using an innovative approach whereby the antigen is expressed in association with extracellular vesicles (EVs) to facilitate antigen cross-presentation and improve induced immunity. Our two groups had independently shown previously that DNA vaccines encoding EV-associated antigens are more efficient at inducing cytotoxic T-cell responses than vaccines encoding the non-EV-associated antigen. Here, we compared our two approaches to associate the ovalbumin (OVA) antigen to EVs: (a) by fusion to the lipid-binding domain C1C2 of MFGE8(=lactadherin), which is exposed on the surface of secreted membrane vesicles; and (b) by fusion to retroviral Gag capsid protein, which is incorporated inside membrane-enclosed virus-like particles. Plasmids encoding either form of modified OVA were used as DNA-based vaccines (i.e. injected into mice to allow in vivo expression of the antigen associated to EVs). We show that both DNA vaccines induced, with similar efficiency, OVA-specific CD8(+) T cells and total IgG antibodies. By contrast, each vaccine preferentially stimulated different isotypes of immunoglobulins, and the OVA-C1C2-encoding vaccine favoured antigen-specific CD4(+) T lymphocyte induction as compared to the Gag-OVA vaccine. Nevertheless, both OVA-C1C2 and Gag-OVA vaccines efficiently prevented in vivo outgrowth of OVA-expressing tumours and reduced tumour progression when administered to tumour-bearing mice, although with variable efficacies depending on the tumour models. DNA vaccines encoding EV-associated antigens are thus promising immunotherapy tools in cancer but also potentially other diseases. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 149 (pelleting) Protein markers EV: OVA non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW32.1 Pelleting: adjusted k-factor 149 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins OVA ELISA Antibody details provided? No Detected EV-associated proteins OVA Characterization: Particle analysis NTA

	EV140281	2/2	Mus musculus	NAY	(d)(U)C Filtration	Sano S	2014	22%
Study summary Full title Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells All authors Sano S, Izumi Y, Yamaguchi T, Yamazaki T, Tanaka M, Shiota M, Osada-Oka M, Nakamura Y, Wei M, Wanibuchi H, Iwao H, Yoshiyama M Journal Biochem Biophys Res Commun Abstract Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with ad (show more...)Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3-4-fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: HSP90/ HSP72/ HSC70 non-EV: Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 180 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP90/ HSC70/ HSP72 ELISA Antibody details provided? No Detected EV-associated proteins HSC70/ HSP72 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140209	1/2	Rattus norvegicus/rattus	NAY	(d)(U)C Filtration	Rodríguez-Suárez E	2014	22%
Study summary Full title Quantitative proteomic analysis of hepatocyte-secreted extracellular vesicles reveals candidate markers for liver toxicity All authors Rodríguez-Suárez E, Gonzalez E, Hughes C, Conde-Vancells J, Rudella A, Royo F, Palomo L, Elortza F, Lu SC, Mato JM, Vissers JP, Falcón-Pérez JM Journal J Proteomics Abstract Extracellular vesicles have created great interest as possible source of biomarkers for different bi (show more...)Extracellular vesicles have created great interest as possible source of biomarkers for different biological processes and diseases. Although the biological function of these vesicles is not fully understood, it is clear that they participate in the removal of unnecessary cellular material and act as carriers of various macromolecules and signals between the cells. In this report, we analyzed the proteome of extracellular vesicles secreted by primary hepatocytes. We used one- and two-dimensional liquid chromatography combined with data-independent mass spectrometry. Employing label-free quantitative proteomics, we detected significant changes in vesicle protein expression levels in this in vitro model after exposure to well-known liver toxins (galactosamine and Escherichia coli-derived lipopolysaccharide). The results allowed us to identify candidate markers for liver injury. We validated a number of these markers in vivo, providing the basis for the development of novel methods to evaluate drug toxicity. This report strongly supports the application of proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems. Analysis of such systems should help to identify specific markers for various biological processes and pathological conditions.BIOLOGICAL SIGNIFICANCE: Identification of low invasive candidate marker for hepatotoxicity. Support to apply proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems to identify low invasive markers for diseases. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 256 (pelleting) Protein markers EV: Alix/ HSP70/ HSP90 non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP90/ HSP70 Characterization: Particle analysis None

	EV140209	2/2	Rattus norvegicus/rattus	Serum	(d)(U)C Filtration	Rodríguez-Suárez E	2014	22%
Study summary Full title Quantitative proteomic analysis of hepatocyte-secreted extracellular vesicles reveals candidate markers for liver toxicity All authors Rodríguez-Suárez E, Gonzalez E, Hughes C, Conde-Vancells J, Rudella A, Royo F, Palomo L, Elortza F, Lu SC, Mato JM, Vissers JP, Falcón-Pérez JM Journal J Proteomics Abstract Extracellular vesicles have created great interest as possible source of biomarkers for different bi (show more...)Extracellular vesicles have created great interest as possible source of biomarkers for different biological processes and diseases. Although the biological function of these vesicles is not fully understood, it is clear that they participate in the removal of unnecessary cellular material and act as carriers of various macromolecules and signals between the cells. In this report, we analyzed the proteome of extracellular vesicles secreted by primary hepatocytes. We used one- and two-dimensional liquid chromatography combined with data-independent mass spectrometry. Employing label-free quantitative proteomics, we detected significant changes in vesicle protein expression levels in this in vitro model after exposure to well-known liver toxins (galactosamine and Escherichia coli-derived lipopolysaccharide). The results allowed us to identify candidate markers for liver injury. We validated a number of these markers in vivo, providing the basis for the development of novel methods to evaluate drug toxicity. This report strongly supports the application of proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems. Analysis of such systems should help to identify specific markers for various biological processes and pathological conditions.BIOLOGICAL SIGNIFICANCE: Identification of low invasive candidate marker for hepatotoxicity. Support to apply proteomics in the study of extracellular vesicles released by well-controlled in vitro cellular systems to identify low invasive markers for diseases. (hide) EV-METRIC 22% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 256 (pelleting) Protein markers EV: Alix/ HSP70/ HSP90 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Rattus norvegicus/rattus Sample Type Serum Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP90/ HSP70 Characterization: Particle analysis None

	EV140132	1/4	Mus musculus	Blood plasma	(d)(U)C	Povero D	2014	22%
Study summary Full title Circulating Extracellular Vesicles with Specific Proteome and Liver MicroRNAs Are Potential Biomarkers for Liver Injury in Experimental Fatty Liver Disease All authors Povero D, Eguchi A, Li H, Johnson CD, Papouchado BG, Wree A, Messer K, Feldstein AE Journal PLoS One Abstract BACKGROUND & AIM: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease (show more...)BACKGROUND & AIM: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. DESIGN: Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. RESULTS: We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver. CONCLUSIONS: These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD. (hide) EV-METRIC 22% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD81/ ASGPR1/ Annexin5/ ICAM1/ Vanin non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ ICAM1/ Annexin5/ Vanin/ ASGPR1 ELISA Antibody details provided? No Detected EV-associated proteins ICAM1/ Annexin5/ Vanin/ ASGPR1 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140091	1/1	Homo sapiens	NAY	(d)(U)C	Phuyal S	2014	22%
Study summary Full title The ether lipid precursor hexadecylglycerol stimulates the release and changes the composition of exosomes derived from PC-3 cells All authors Phuyal S, Skotland T, Hessvik NP, Simolin H, Øverbye A, Brech A, Parton RG, Ekroos K, Sandvig K, Llorente A Journal J Biol Chem Abstract Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membra (show more...)Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ Flotilin1/ Alix/ Annexin2/ Caveolin1/ CD9 non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ CD9/ Flotilin1/ TSG101/ Caveolin1/ Annexin2 ELISA Antibody details provided? No Detected EV-associated proteins Caveolin1/ Annexin2 Characterization: Particle analysis NTA EM EM-type immune EM EM protein CD63 Image type Wide-field

	EV140206	1/1	Homo sapiens	NAY	(d)(U)C	Phuyal S	2014	22%
Study summary Full title Regulation of exosome release by glycosphingolipids and flotillins All authors Phuyal S, Hessvik NP, Skotland T, Sandvig K, Llorente A Journal FEBS J Abstract Exosomes are released by cells after fusion of multivesicular bodies with the plasma membrane. The m (show more...)Exosomes are released by cells after fusion of multivesicular bodies with the plasma membrane. The molecular mechanism of this process is still unclear. We investigated the role of sphingolipids and flotillins, which constitute a raft-associated family of proteins, in the release of exosomes. Interestingly, our results show that dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase, seemed to affect the composition of exosomes released from PC-3 cells. However, the inhibition of ceramide formation from the de novo pathway by fumonisin B1 did not affect exosome secretion. Moreover, in contrast to findings obtained with other cell lines published so far, inhibition of neutral sphingomyelinase 2, an enzyme that catalyzes the formation of ceramide from sphingomyelin, did not inhibit the secretion of exosomes in PC-3 cells. Finally, small interfering RNA-mediated downregulation of flotillin-1 and flotillin-2 did not significantly change the levels of released exosomes as such, but seemed to affect the composition of exosomes. In conclusion, our results reveal the involvement of glycosphingolipids and flotillins in the release of exosomes from PC-3 cells, and indicate that the role of ceramide in exosome formation may be cell-dependent. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 95.8 (pelleting) / 95.8 (washing) Protein markers EV: TSG101/ CD63/ Vinculin/ Flotilin1/ Alix/ CD81/ Annexin2/ Flotillin2/ Caveolin1/ CD9 non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type MLA80 Pelleting: adjusted k-factor 95.8 Wash: Rotor Type MLA80 Wash: adjusted k-factor 95.8 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ CD81/ CD9/ Flotilin1/ TSG101/ Caveolin1/ Annexin2/ Vinculin/ Flotillin2 ELISA Antibody details provided? No Detected EV-associated proteins Caveolin1/ Annexin2/ Vinculin/ Flotillin2 Characterization: Particle analysis NTA

	EV140202	1/1	Homo sapiens	NAY	(d)(U)C DG Filtration	Ota M	2014	22%
Study summary Full title MHC class I molecules are incorporated into human herpesvirus-6 viral particles and released into the extracellular environment All authors Ota M, Serada S, Naka T, Mori Y Journal Microbiol Immunol Abstract Human herpesvirus-6 (HHV-6), which belongs to the betaherpesvirus subfamily, mainly replicates in T (show more...)Human herpesvirus-6 (HHV-6), which belongs to the betaherpesvirus subfamily, mainly replicates in T lymphocytes. Here, we show that MHC class I molecules are incorporated into HHV-6 viral particles and released into the extracellular environment. In addition, HHV-6A/B-infected T cells showed reduced surface and intracellular expression of MHC class I molecules. The cellular machinery responsible for molecular transport appears to be modified upon HHV-6 infection, causing MHC class I molecules to be transported to virion assembly sites. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: CD63/ MHC1 non-EV: Proteomics yes Show all info Study aim Other/Virus function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ MHC1 ELISA Antibody details provided? No Detected EV-associated proteins MHC1 Characterization: Particle analysis EM EM-type immune EM EM protein MHC1 Image type Wide-field

	EV140377	1/2	Homo sapiens	NAY	(d)(U)C Filtration	Ogata-Kawata H	2014	22%
Study summary Full title Circulating exosomal microRNAs as biomarkers of colon cancer All authors Ogata-Kawata H, Izumiya M, Kurioka D, Honma Y, Yamada Y, Furuta K, Gunji T, Ohta H, Okamoto H, Sonoda H, Watanabe M, Nakagama H, Yokota J, Kohno T, Tsuchiya N Journal PLoS One Abstract PURPOSE: Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic bio (show more...)PURPOSE: Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined. EXPERIMENTAL DESIGN: Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients. RESULTS: The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis. CONCLUSION: Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 54.74 (pelleting) Protein markers EV: CD81 non-EV: Beta-tubulin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type TLA110 Pelleting: adjusted k-factor 54.74 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81 Detected contaminants Beta-tubulin Characterization: Particle analysis None

	EV140377	2/2	Homo sapiens	Serum	(d)(U)C Filtration	Ogata-Kawata H	2014	22%
Study summary Full title Circulating exosomal microRNAs as biomarkers of colon cancer All authors Ogata-Kawata H, Izumiya M, Kurioka D, Honma Y, Yamada Y, Furuta K, Gunji T, Ohta H, Okamoto H, Sonoda H, Watanabe M, Nakagama H, Yokota J, Kohno T, Tsuchiya N Journal PLoS One Abstract PURPOSE: Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic bio (show more...)PURPOSE: Exosomal microRNAs (miRNAs) have been attracting major interest as potential diagnostic biomarkers of cancer. The aim of this study was to characterize the miRNA profiles of serum exosomes and to identify those that are altered in colorectal cancer (CRC). To evaluate their use as diagnostic biomarkers, the relationship between specific exosomal miRNA levels and pathological changes of patients, including disease stage and tumor resection, was examined. EXPERIMENTAL DESIGN: Microarray analyses of miRNAs in exosome-enriched fractions of serum samples from 88 primary CRC patients and 11 healthy controls were performed. The expression levels of miRNAs in the culture medium of five colon cancer cell lines were also compared with those in the culture medium of a normal colon-derived cell line. The expression profiles of miRNAs that were differentially expressed between CRC and control sample sets were verified using 29 paired samples from post-tumor resection patients. The sensitivities of selected miRNAs as biomarkers of CRC were evaluated and compared with those of known tumor markers (CA19-9 and CEA) using a receiver operating characteristic analysis. The expression levels of selected miRNAs were also validated by quantitative real-time RT-PCR analyses of an independent set of 13 CRC patients. RESULTS: The serum exosomal levels of seven miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) were significantly higher in primary CRC patients, even those with early stage disease, than in healthy controls, and were significantly down-regulated after surgical resection of tumors. These miRNAs were also secreted at significantly higher levels by colon cancer cell lines than by a normal colon-derived cell line. The high sensitivities of the seven selected exosomal miRNAs were confirmed by a receiver operating characteristic analysis. CONCLUSION: Exosomal miRNA signatures appear to mirror pathological changes of CRC patients and several miRNAs are promising biomarkers for non-invasive diagnosis of the disease. (hide) EV-METRIC 22% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 54.74 (pelleting) Protein markers EV: CD81 non-EV: Beta-tubulin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type TLA110 Pelleting: adjusted k-factor 54.74 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81 Detected contaminants Beta-tubulin Characterization: Particle analysis None

	EV140197	1/1	Mus musculus	NAY	(d)(U)C	Merezhko M	2014	22%
Study summary Full title Multiplex assay for live-cell monitoring of cellular fates of amyloid-beta precursor protein (APP) All authors Merezhko M, Muggalla P, Nykänen NP, Yan X, Sakha P, Huttunen HJ Journal PLoS One Abstract Amyloid-? precursor protein (APP) plays a central role in pathogenesis of Alzheimer's disease. APP h (show more...)Amyloid-? precursor protein (APP) plays a central role in pathogenesis of Alzheimer's disease. APP has a short half-life and undergoes complex proteolytic processing that is highly responsive to various stimuli such as changes in cellular lipid or energy homeostasis. Cellular trafficking of APP is controlled by its large protein interactome, including dozens of cytosolic adaptor proteins, and also by interactions with lipids. Currently, cellular regulation of APP is mostly studied based on appearance of APP-derived proteolytic fragments to conditioned media and cellular extracts. Here, we have developed a novel live-cell assay system based on several indirect measures that reflect altered APP trafficking and processing in cells. Protein-fragment complementation assay technology for detection of APP-BACE1 protein-protein interaction forms the core of the new assay. In a multiplex form, the assay can measure four endpoints: total cellular APP level, total secreted sAPP level in media, APP-BACE1 interaction in cells and in exosomes released by the cells. Functional validation of the assay with pharmacological and genetic tools revealed distinct patterns of cellular fates of APP, with immediate mechanistic implications. This new technology will facilitate functional genomics studies of late-onset Alzheimer's disease, drug discovery efforts targeting APP and characterization of the physiological functions of APP and its proteolytic fragments. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 255.8 (pelleting) / 255.8 (washing) Protein markers EV: Alix non-EV: Proteomics no Show all info Study aim Other/characterization of cellular regulation of amyloid precursor protein Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Wash: volume per pellet (ml) 1 Wash: Rotor Type SW41 Wash: adjusted k-factor 255.8 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix Characterization: Particle analysis None

	EV140196	1/1	Homo sapiens	Blood plasma	(d)(U)C	Martinez-Pinna R	2014	22%
Study summary Full title Label-free quantitative proteomic analysis of human plasma-derived microvesicles to find protein signatures of abdominal aortic aneurysms All authors Martinez-Pinna R, Gonzalez de Peredo A, Monsarrat B, Burlet-Schiltz O, Martin-Ventura JL Journal Proteomics Clin Appl Abstract PURPOSE: To find potential biomarkers of abdominal aortic aneurysms (AAA), we performed a differenti (show more...)PURPOSE: To find potential biomarkers of abdominal aortic aneurysms (AAA), we performed a differential proteomic study based on human plasma-derived microvesicles. EXPERIMENTAL DESIGN: Exosomes and microparticles isolated from plasma of AAA patients and control subjects (n = 10 each group) were analyzed by a label-free quantitative MS-based strategy. Homemade and publicly available software packages have been used for MS data analysis. RESULTS: The application of two kinds of bioinformatic tools allowed us to find differential protein profiles from AAA patients. Some of these proteins found by the two analysis methods belong to main pathological mechanisms of AAA such as oxidative stress, immune-inflammation, and thrombosis. CONCLUSIONS AND CLINICAL RELEVANCE: Data analysis from label-free MS-based experiments requires the use of sophisticated bioinformatic approaches to perform quantitative studies from complex protein mixtures. The application of two of these bioinformatic tools provided us a preliminary list of differential proteins found in plasma-derived microvesicles not previously associated to AAA, which could help us to understand the pathological mechanisms related to this disease. (hide) EV-METRIC 22% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Clathrin non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Clathrin ELISA Antibody details provided? No Detected EV-associated proteins Clathrin Characterization: Particle analysis None

	EV140195	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Mahmoodzadeh Hosseini H	2014	22%
Study summary Full title Exosome/staphylococcal enterotoxin B, an anti tumor compound against pancreatic cancer All authors Mahmoodzadeh Hosseini H, Ali Imani Fooladi A, Soleimanirad J, Reza Nourani M, Mahdavi M Journal J BUON Abstract PURPOSE: Exosomes (EXOs) are acellular vehicles used for cancer immunotherapy due to their immune-in (show more...)PURPOSE: Exosomes (EXOs) are acellular vehicles used for cancer immunotherapy due to their immune-inducing properties. We synthesized a novel structure based on EXOs and staphylococcal enterotoxin B (SEB) and surveyed its cytostatic effect on a pancreatic cell line. METHODS: EXOs were purified from tumor cells and SEB was anchored on it by protein transfer method. To determine the cytotoxic and apoptosis-inducing effect of this structure, treated cells with different concentrations of EXO/SEB were examined by MTT assay and Hoechst staining method. In addition, the expression rate of bcl-2, bax, bak, fas, bcl-xl and the activity of caspase-3 and caspase-9 were assessed. RESULTS: We observed that 0.5 and 2.5 ?g/100?l of EXO/ SEB significantly (p<0.001) stimulated apoptosis after 24 hrs. The concentrations of 0.5 and 2.5?g/100?l of EXO/SEB raised the expression rate of bax, bak, fas (p<0.001) but had no impact on bcl-2 and bcl-xl after 48 hrs. Furthermore, it was shown that 0.5, 2.5 and 5 ?g/100?l of EXO/SEB only increased the activity of caspase-3 after 48 hrs (p<0.001). CONCLUSION: Our designed structure, the EXO/SEB, is a novel model being able to induce apoptosis. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 25.32 (pelleting) Protein markers EV: HSP70 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Pelleting: rotor type TLA100 Pelleting: adjusted k-factor 25.32 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140052	2/2	Homo sapiens	Serum	(d)(U)C DC Filtration	Lundholm M	2014	22%
Study summary Full title Prostate tumor-derived exosomes down-regulate NKG2D expression on natural killer cells and CD8+ T cells: mechanism of immune evasion All authors Lundholm M, Schröder M, Nagaeva O, Baranov V, Widmark A, Mincheva-Nilsson L, Wikström P Journal PLoS One Abstract Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have e (show more...)Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8+ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8+ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8+ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape. (hide) EV-METRIC 22% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration Protein markers EV: PSMA/ CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ PSMA Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins PSMA Characterization: Particle analysis None

	EV140190	1/1	Homo sapiens	NAY	(d)(U)C	Lopatina T	2014	22%
Study summary Full title Platelet-derived growth factor regulates the secretion of extracellular vesicles by adipose mesenchymal stem cells and enhances their angiogenic potential All authors Lopatina T, Bruno S, Tetta C, Kalinina N, Porta M, Camussi G Journal Cell Commun Signal Abstract BACKGROUND: Several studies demonstrate the role of adipose mesenchymal stem cells (ASCs) in angioge (show more...)BACKGROUND: Several studies demonstrate the role of adipose mesenchymal stem cells (ASCs) in angiogenesis. The angiogenic mechanism has been ascribed to paracrine factors since these cells secrete a plenty of signal molecules and growth factors. Recently it has been suggested that besides soluble factors, extracellular vesicles (EVs) that include exosomes and microvesicles may play a major role in cell-to-cell communication. It has been shown that EVs are implicated in the angiogenic process. RESULTS: Herein we studied whether EVs released by ASCs may mediate the angiogenic activity of these cells. Our results demonstrated that ASC-derived EVs induced in vitro vessel-like structure formation by human microvascular endothelial cells (HMEC). EV-stimulated HMEC when injected subcutaneously within Matrigel in SCID mice formed vessels. Treatment of ASCs with platelet-derived growth factor (PDGF) stimulated the secretion of EVs, changed their protein composition and enhanced the angiogenic potential. At variance of EVs released in basal conditions, PDGF-EVs carried c-kit and SCF that played a role in angiogenesis as specific blocking antibodies inhibited in vitro vessel-like structure formation. The enhanced content of matrix metalloproteinases in PDGF-EVs may also account for their angiogenic activity. CONCLUSIONS: Our findings indicate that EVs released by ASCs may contribute to the ASC-induced angiogenesis and suggest that PDGF may trigger the release of EVs with an enhanced angiogenic potential. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD81/ CD63 non-EV: SCF/ PDGF/ c-kit Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 180 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ CD81 Detected contaminants c-kit/ SCF/ PDGF Characterization: Particle analysis NTA

	EV140085	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Liu T	2014	22%
Study summary Full title Functional prostate-specific membrane antigen is enriched in exosomes from prostate cancer cells All authors Liu T, Mendes DE, Berkman CE Journal Int J Oncol Abstract Developing simple and effective approaches to detect tumor markers will be critical for early diagno (show more...)Developing simple and effective approaches to detect tumor markers will be critical for early diagnosis or prognostic evaluation of prostate cancer treatment. Prostate?specific membrane antigen (PSMA) has been validated as an important tumor marker for prostate cancer progression including angiogenesis and metastasis. As a type II membrane protein, PSMA can be constitutively internalized from the cell surface into endosomes. Early endosomes can fuse with multivesicular bodies (MVB) to form and secrete exosomes (40-100 nm) into the extracellular environment. Herein, we tested whether some of the endosomal PSMA could be transferred to exosomes as an extracellular resource for PSMA. Using PSMA-positive LNCaP cells, the secreted exosomes were collected and isolated from the cultured media. The vesicular structures of exosomes were identified by electron microscopy, and exosomal marker protein CD9 and tumor susceptibility gene (TSG 101) were confirmed by western blot analysis. Our present data demonstrate that PSMA can be enriched in exosomes, exhibiting a higher content of glycosylation and partial proteolysis in comparison to cellular PSMA. An in vitro enzyme assay further confirmed that exosomal PSMA retains functional enzymatic activity. Therefore, our data may suggest a new role for PSMA in prostate cancer progression, and provide opportunities for developing non-invasive approaches for diagnosis or prognosis of prostate cancer. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: TSG101/ PSMA/ GAPDH/ Alpha-tubulin/ EpCAM/ CD9 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Wash: volume per pellet (ml) 10 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101/ EpCAM/ PSMA/ GAPDH/ Alpha-tubulin ELISA Antibody details provided? No Detected EV-associated proteins EpCAM/ PSMA/ GAPDH/ Alpha-tubulin Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140188	1/1	Homo sapiens	NAY	(d)(U)C	Liang Y	2014	22%
Study summary Full title Complex N-linked glycans serve as a determinant for exosome/microvesicle cargo recruitment All authors Liang Y, Eng WS, Colquhoun DR, Dinglasan RR, Graham DR, Mahal LK Journal J Biol Chem Abstract Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from near (show more...)Exosomes, also known as microvesicles (EMVs), are nano-sized membranous particles secreted from nearly all mammalian cell types. These nanoparticles play critical roles in many physiological processes including cell-cell signaling, immune activation, and suppression and are associated with disease states such as tumor progression. The biological functions of EMVs are highly dependent on their protein composition, which can dictate pathogenicity. Although some mechanisms have been proposed for the regulation of EMV protein trafficking, little attention has been paid to N-linked glycosylation as a potential sorting signal. Previous work from our laboratory found a conserved glycan signature for EMVs, which differed from that of the parent cell membranes, suggesting a potential role for glycosylation in EMV biogenesis. In this study, we further explore the role of glycosylation in EMV protein trafficking. We identify EMV glycoproteins and demonstrate alteration of their recruitment as a function of their glycosylation status upon pharmacological manipulation. Furthermore, we show that genetic manipulation of the glycosylation levels of a specific EMV glycoprotein, EWI-2, directly impacts its recruitment as a function of N-linked glycan sites. Taken together, our data provide strong evidence that N-linked glycosylation directs glycoprotein sorting into EMVs. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes / microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: ADAM10/ CD63/ G3BP/ CD81 non-EV: Proteomics yes Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ ADAM10/ G3BP ELISA Antibody details provided? No Detected EV-associated proteins ADAM10/ G3BP Characterization: Particle analysis None

	EV140186	1/1	Homo sapiens	NAY	(d)(U)C	Li J	2014	22%
Study summary Full title beta-Elemene against human lung cancer via up-regulation of P53 protein expression to promote the release of exosome All authors Li J, JunYu, Liu A, Wang Y Journal Lung Cancer Abstract BACKGROUND: ?-Elemene, a novel antitumor plant drug extracted from the traditional Chinese medicinal (show more...)BACKGROUND: ?-Elemene, a novel antitumor plant drug extracted from the traditional Chinese medicinal herb Zedoary, has been shown to be effective against a wide variety of tumors. Recent studies have indicated that ?-elemene can inhibit the growth of lung cancer cells; however, the exact mechanism of ?-element's action in lung cancer remains largely unknown. In the present study, the antitumor effect of ?-elemene on human lung cancer cells and the mechanism involved has been investigated. METHODS: The inhibitory effects of ?-elemene on cell growth were measured by Trypan Blue exclusion and MTT assay. Flow cytometric analysis was used to detect the cells' apoptotic rate. The expression of P53 mRNA and protein were measured by RT-PCR and Western blot analysis, respectively. Exosomes were isolated by differential centrifugation steps and analyzed by electron microscopy and western blotting. P53 knockdown cells were established through transfection with P53 siRNA. To investigate the effect of ?-elemene on the tumor growth in vivo, a Xenograft nude mouse model was established by injecting the A549 cells into the back of a BABL/c nude mouse. RESULTS: ?-Elemene markedly inhibited growth and induced apoptosis in lung cancer cells. The levels of the anti-apoptotic genes Bcl-2 and Bcl-xl in A549 cells decreased, while expression of P53 and production of exosomes increased after ?-elemene treatment. Further siRNA studies suggested that the effect of ?-elemene on A549 cells is dependent on P53 expression. Exosomes derived from A549 cultured with a human lung cancer cell line exhibited decreased tumor cell proliferation. The in vivo study demonstrated that ?-elemene inhibited tumor growth, and up-regulated the expression of P53 and the release of exosome. CONCLUSION: Our results demonstrated ?-elemene acts on lung cancer cells in a P53 dependent manner and exosomes are involved in the regulation of cell proliferation. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 248.9 (pelleting) Protein markers EV: HSP70/ Beta-actin/ Flotilin1/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Pelleting: rotor type KAL40.100 Pelleting: adjusted k-factor 248.9 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotilin1/ HSP70/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis None

	EV140360	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Lee HD	2014	22%
Study summary Full title Ectodomain of ADAM15 is shed from secretory exosomes All authors Lee HD, Kim YH, Koo BH, Kim DS Journal BMB Rep Abstract We demonstrated previously that a disintegrin and metalloproteinase 15 (ADAM15) is released into the (show more...)We demonstrated previously that a disintegrin and metalloproteinase 15 (ADAM15) is released into the extracellular space as an exosomal component, and that ADAM15-rich exosomes have tumor suppressive functions. However, the suppressive mechanism of ADAM15-rich exosomes remains unclear. In this study, we show that the ADAM15 ectodomain is cleaved from released exosomes. This shedding process of the ADAM15 ectodomain was dramatically enhanced in conditioned ovarian cancer cell medium. Proteolytic cleavage was completely blocked by phenylmethylsulfonyl fluoride, indicating that a serine protease is responsible for exosomal ADAM15 shedding. Experimental evidence indicates that the ADAM15 ectodomain itself has comparable functions with those of ADAM15-rich exosomes, which effectively inhibit vitronectininduced cancer cell migration and activation of the MEK/extracellular regulated kinase signaling pathway. We present a tumor suppressive mechanism for ADAM15 exosomes and provide insight into the functional significance of exosomes that generate tumor-inhibitory factors. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 138.6 (pelleting) Protein markers EV: TSG101/ GAPDH/ ADAM15 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW55 Pelleting: adjusted k-factor 138.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ GAPDH/ ADAM15 ELISA Antibody details provided? No Detected EV-associated proteins GAPDH/ ADAM15 Characterization: Particle analysis None

	EV140185	1/2	Canis familiaris	NAY	(d)(U)C	Kwon SH	2014	22%
Study summary Full title Intercellular transfer of GPRC5B via exosomes drives HGF-mediated outward growth All authors Kwon SH, Liu KD, Mostov KE Journal Curr Biol Abstract How cells communicate during development and regeneration is a critical question. One mechanism of i (show more...)How cells communicate during development and regeneration is a critical question. One mechanism of intercellular communication is via exosomes, extracellular vesicles that originate by the fusion of multivesicular endosomes with the plasma membrane [1-8]. To model exosome-based intercellular communication, we used Madin-Darby canine kidney (MDCK) cell cysts grown in 3D gels of extracellular matrix, which form tubules in response to hepatocyte growth factor (HGF). We report that GPRC5B, an orphan G protein coupled receptor, is in exosomes produced by HGF-treated cysts and released into the cyst lumen. Exosomal GPRC5B is taken up by nearby cells and together with HGF promotes extracellular signal-regulated kinase 1/2 (ERK1/2) activation and tubulogenesis, even under conditions where tubulogenesis would otherwise not occur. Recovery from injury, such as acute kidney injury (AKI), often recapitulates developmental processes. Here, we show that GPRC5B is elevated in urinary exosomes from patients with AKI. Our results elucidate how GPRC5B is carried by exosomes and augments HGF-induced morphogenesis. The unexpected role of exosomes in transporting GPRC5B between cells during morphogenesis and the ability of GPRC5B to predict the disease state of AKI elucidate a novel mechanism for intercellular communication during development and repair. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Canis familiaris Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 75 Characterization: Protein analysis Western Blot Antibody details provided? No Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140357	1/1	Homo sapiens	NAY	(d)(U)C DC Filtration	Kruger S	2014	22%
Study summary Full title Molecular characterization of exosome-like vesicles from breast cancer cells All authors Kruger S, Abd Elmageed ZY, Hawke DH, Wörner PM, Jansen DA, Abdel-Mageed AB, Alt EU, Izadpanah R Journal BMC Cancer Abstract BACKGROUND: Membrane vesicles released by neoplastic cells into extracellular medium contain potenti (show more...)BACKGROUND: Membrane vesicles released by neoplastic cells into extracellular medium contain potential of carrying arrays of oncogenic molecules including proteins and microRNAs (miRNA). Extracellular (exosome-like) vesicles play a major role in cell-to-cell communication. Thus, the characterization of proteins and miRNAs of exosome-like vesicles is imperative in clarifying intercellular signaling as well as identifying disease markers. METHODS: Exosome-like vesicles were isolated using gradient centrifugation from MCF-7 and MDA-MB 231 cultures. Proteomic profiling of vesicles using liquid chromatography-mass spectrometry (LC-MS/MS) revealed different protein profiles of exosome-like vesicles derived from MCF-7 cells (MCF-Exo) than those from MDA-MB 231 cells (MDA-Exo). RESULTS: The protein database search has identified 88 proteins in MDA-Exo and 59 proteins from MCF-Exo. Analysis showed that among all, 27 proteins were common between the two exosome-like vesicle types. Additionally, MDA-Exo contains a higher amount of matrix-metalloproteinases, which might be linked to the enhanced metastatic property of MDA-MB 231 cells. In addition, microarray analysis identified several oncogenic miRNA between the two types vesicles. CONCLUSIONS: Identification of the oncogenic factors in exosome-like vesicles is important since such vesicles could convey signals to non-malignant cells and could have an implication in tumor progression and metastasis. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Exosome-like vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration Adj. k-factor 255.8 (pelleting) Protein markers EV: Annexin2/ Alpha-enolase non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Annexin2/ Alpha-enolase ELISA Antibody details provided? No Detected EV-associated proteins Annexin2/ Alpha-enolase Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140183	1/1	Homo sapiens	NAY	(d)(U)C	Kore RA	2014	22%
Study summary Full title Inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha, upregulated in glioblastoma multiforme, raise the levels of CRYAB in exosomes secreted by U373 glioma cells All authors Kore RA, Abraham EC Journal Biochem Biophys Res Commun Abstract In the brain, levels of inflammatory cytokines, interleukin-1 beta (IL-1?) and tumor necrosis factor (show more...)In the brain, levels of inflammatory cytokines, interleukin-1 beta (IL-1?) and tumor necrosis factor-alpha (TNF-?), are elevated under traumatic brain injury, neuroinflammatory conditions and glioblastoma multiforme (GBM). In GBM, the levels of small heat shock protein, CRYAB (HspB5) are also reported to be elevated, where it has been shown to exert anti-apoptotic activity. Interestingly, CRYAB is secreted via exosomes by various cells. In order to understand the relation between inflammatory cytokines and CRYAB, U373 glioma cells, were stimulated with proinflammatory cytokines, IL-1? and TNF-?, and their effect on CRYAB levels in cells and secreted exosomes was studied. Our results show that U373 cells produce and secrete CRYAB via exosomes and that stimulation with IL-1? and TNF-? significantly increase the levels of CRYAB in not only the cells but also in the secreted exosomes. In addition, cytokine stimulation of U373 cells brings about changes in the secreted exosomal proteome, many of which are involved in cancer progression. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Beta-actin/ CD63/ CD9 non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 240 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140176	1/2	Homo sapiens	NAY	(d)(U)C	Kato T	2014	22%
Study summary Full title Exosomes from IL-1beta stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes All authors Kato T, Miyaki S, Ishitobi H, Nakamura Y, Nakasa T, Lotz MK, Ochi M Journal Arthritis Res Ther Abstract INTRODUCTION: Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degrada (show more...)INTRODUCTION: Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells. METHODS: Human synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1? (IL-1?) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis. RESULTS: Exosomes from IL-1? stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1? stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1?, TNF? MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1? stimulated SFB compared to non-stimulated SFB. CONCLUSIONS: Exosomes from IL-1? stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ Flotilin1/ CD9 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ CD9/ Flotilin1 Characterization: Particle analysis TRPS

	EV140082	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Kambe S	2014	22%
Study summary Full title Human Exosomal Placenta-Associated miR-517a-3p Modulates the Expression of PRKG1 mRNA in Jurkat Cells All authors Kambe S, Yoshitake H, Yuge K, Ishida Y, Ali MM, Takizawa T, Kuwata T, Ohkuchi A, Matsubara S, Suzuki M, Takeshita T, Saito S, Takizawa T Journal Biol Reprod Abstract During pregnancy, human placenta-associated microRNAs (miRNAs) derived from the miRNA cluster in hum (show more...)During pregnancy, human placenta-associated microRNAs (miRNAs) derived from the miRNA cluster in human chromosome 19 are expressed in villous trophoblasts and secreted into maternal circulation via exosomes; however, little is known about whether circulating placenta-associated miRNAs are transferred into maternal immune cells via exosomes, and modulate expression of target genes in the recipient cells. We employed an in vitro model of trophoblast-immune cell communication using BeWo cells (a human trophoblast cell line) and Jurkat cells (a human leukemic T-cell line) and investigated whether BeWo exosomal placenta-associated miRNAs can suppress expression of target genes in the recipient Jurkat cells. Using this system, we identified PRKG1 as a target gene of placenta-associated miRNA miR-517a-3p. Moreover, we demonstrated that BeWo exosomal miR-517a-3p was internalized into Jurkat cells and subsequently suppressed the expression of PRKG1 in recipient Jurkat cells. Furthermore, using peripheral blood natural killer (NK) cells in vivo, we confirmed that circulating miR-517a-3p was delivered into maternal NK cells as it was into Jurkat cells in vitro. Placenta-associated miR-517a-3p was incorporated into maternal NK cells in the third trimester, and it was rapidly cleared after delivery. Expression levels of miR-517a-3p and its target mRNA PRKG1 were inversely correlated in NK cells before and after delivery. These in vitro and in vivo results suggest that exosome-mediated transfer of placenta-associated miRNAs and subsequent modulation of their target genes occur in maternal NK cells. The present study provides novel insight into our understanding of placenta-maternal communication. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ TSG101/ CD63 non-EV: Proteomics no TEM measurements 85+-33 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101 Characterization: Particle analysis EM EM-type immune EM EM protein CD63 Image type Close-up Report size (nm) 85+-33

	EV140173	1/1	Homo sapiens	NAY	(d)(U)C DG	Kalamvoki M	2014	22%
Study summary Full title Cells infected with herpes simplex virus 1 export to uninfected cells exosomes containing STING, viral mRNAs, and microRNAs All authors Kalamvoki M, Du T, Roizman B Journal Proc Natl Acad Sci U S A Abstract STING (stimulator of IFN genes) activates the IFN-dependent innate immune response to infection on s (show more...)STING (stimulator of IFN genes) activates the IFN-dependent innate immune response to infection on sensing the presence of DNA in cytosol. The quantity of STING accumulating in cultured cells varies; it is relatively high in some cell lines [e.g., HEp-2, human embryonic lung fibroblasts (HEL), and HeLa] and low in others (e.g., Vero cells). In a preceding publication we reported that STING was stable in four cell lines infected with herpes simplex virus 1 and that it was actively stabilized in at least two cell lines derived from human cancers. In this report we show that STING is exported from HEp-2 cells to Vero cells along with virions, viral mRNAs, microRNAs, and the exosome marker protein CD9. The virions and exosomes copurified. The quantity of STING and CD9 exported from one cell line to another was inoculum-size-dependent and reflected the levels of STING and CD9 accumulating in the cells in which the virus inoculum was made. The export of STING, an innate immune sensor, and of viral mRNAs whose major role may be in silencing viral genes in latently infected neurons, suggests that the virus has evolved mechanisms that curtail rather than foster the spread of infection under certain conditions. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Adj. k-factor 362.8 (pelleting) Protein markers EV: CD9 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 362.8 Density gradient Lowest density fraction 1.04g/cm3 Highest density fraction 1.09g/cm3 Orientation Top-down Speed (g) 75000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9 Characterization: Particle analysis None

	EV140172	1/2	Homo sapiens	Blood plasma	(d)(U)C	Jiang ZG	2014	22%
Study summary Full title Characterization of circulating microparticle-associated CD39 family ecto-nucleotidases in human plasma All authors Jiang ZG, Wu Y, Csizmadia E, Feldbrügge L, Enjyoji K, Tigges J, Toxavidis V, Stephan H, Müller CE, McKnight CJ, Moss A, Robson SC Journal Purinergic Signal Abstract Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that reg (show more...)Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either -2 or/and -3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism. (hide) EV-METRIC 22% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles microparticles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: non-EV: NTPD8/ CD39/ NTPD2/ NTPD3/ Annexin5 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Characterization: Protein analysis Western Blot Antibody details provided? No Detected contaminants "CD39/ NTPD2/ NTPD3/ NTPD8/ Annexin5" Characterization: Particle analysis

	EV140165	1/2	Homo sapiens	NAY	(d)(U)C Filtration IAF SEC	Hong CS	2014	22%
Study summary Full title Isolation and characterization of CD34+ blast-derived exosomes in acute myeloid leukemia All authors Hong CS, Muller L, Boyiadzis M, Whiteside TL Journal PLoS One Abstract Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected (show more...)Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected at diagnosis contains elevated exosome levels relative to normal donor (ND) plasma. The molecular profile of AML exosomes changes in the course of therapy and may serve as a measure of disease progression or response to therapy. However, plasma contains a mix of exosomes derived from various cell types. To be able to utilize blast-derived exosomes as biomarkers for AML, we have developed an immunoaffinity-based capture method utilizing magnetic microbeads coated with anti-CD34 antibody (Ab). This Ab is specific for CD34, a unique marker of AML blasts. The capture procedure was developed using CD34+ exosomes derived from Kasumi-1 AML cell culture supernatants. The capture capacity of CD34microbeads was shown to linearly correlate with the input exosomes. A 10 uL aliquot of CD34 microbeads was able to capture all of CD34+ exosomes present in 100-1,000 uL of AML plasma. The levels of immunocaptured CD34+ exosomes correlated with the percentages of CD34+ blasts in the AML patients' peripheral blood. The immunocaptured exosomes had a typical cup-shaped morphology by transmission electron microscopy, and their molecular cargo was similar to that of parental blasts. These exosomes were biologically-active. Upon co-incubation with natural killer (NK) cells, captured blast-derived exosomes down-regulated surface NKG2D expression, while non-captured exosomes reduced expression levels of NKp46. Our data provide a proof-of-principle that blast-derived exosomes can be quantitatively recovered from AML patients' plasma, their molecular profile recapitulates that of autologous blasts and they retain the ability to mediate immune suppression. These data suggest that immunocaptured blast-derived exosomes might be useful in diagnosis and/or prognosis of AML in the future. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration IAF SEC Protein markers EV: CD81 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) CD34 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD81 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140165	2/2	Homo sapiens	Blood plasma	(d)(U)C Filtration IAF SEC	Hong CS	2014	22%
Study summary Full title Isolation and characterization of CD34+ blast-derived exosomes in acute myeloid leukemia All authors Hong CS, Muller L, Boyiadzis M, Whiteside TL Journal PLoS One Abstract Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected (show more...)Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected at diagnosis contains elevated exosome levels relative to normal donor (ND) plasma. The molecular profile of AML exosomes changes in the course of therapy and may serve as a measure of disease progression or response to therapy. However, plasma contains a mix of exosomes derived from various cell types. To be able to utilize blast-derived exosomes as biomarkers for AML, we have developed an immunoaffinity-based capture method utilizing magnetic microbeads coated with anti-CD34 antibody (Ab). This Ab is specific for CD34, a unique marker of AML blasts. The capture procedure was developed using CD34+ exosomes derived from Kasumi-1 AML cell culture supernatants. The capture capacity of CD34microbeads was shown to linearly correlate with the input exosomes. A 10 uL aliquot of CD34 microbeads was able to capture all of CD34+ exosomes present in 100-1,000 uL of AML plasma. The levels of immunocaptured CD34+ exosomes correlated with the percentages of CD34+ blasts in the AML patients' peripheral blood. The immunocaptured exosomes had a typical cup-shaped morphology by transmission electron microscopy, and their molecular cargo was similar to that of parental blasts. These exosomes were biologically-active. Upon co-incubation with natural killer (NK) cells, captured blast-derived exosomes down-regulated surface NKG2D expression, while non-captured exosomes reduced expression levels of NKp46. Our data provide a proof-of-principle that blast-derived exosomes can be quantitatively recovered from AML patients' plasma, their molecular profile recapitulates that of autologous blasts and they retain the ability to mediate immune suppression. These data suggest that immunocaptured blast-derived exosomes might be useful in diagnosis and/or prognosis of AML in the future. (hide) EV-METRIC 22% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration IAF SEC Protein markers EV: CD81/ CD63/ GAPDH non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) CD34 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ GAPDH ELISA Antibody details provided? No Detected EV-associated proteins GAPDH Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140117	1/8	Homo sapiens	NAY	(d)(U)C	He M	2014	22%
Study summary Full title Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology All authors He M, Crow J, Roth M, Zeng Y, Godwin AK Journal Lab Chip Abstract Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy (show more...)Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy is difficult, costly, and sometimes not even an option. Tumor-derived exosomes have attracted increasing interest in non-invasive cancer diagnosis and monitoring of treatment response. However, the biology and clinical value of exosomes remains largely unknown due in part to current technical challenges in rapid isolation, molecular classification and comprehensive analysis of exosomes. Here we developed a new microfluidic approach to streamline and expedite the exosome analysis pipeline by integrating specific immunoisolation and targeted protein analysis of circulating exosomes. Compared to the conventional methods, our approach enables selective subpopulation isolation and quantitative detection of surface and intravesicular biomarkers directly from a minimally invasive amount of plasma samples (30 ?L) within ~100 min with markedly improved detection sensitivity. Using this device, we demonstrated phenotyping of exosome subpopulations by targeting a panel of common exosomal and tumor-specific markers and multiparameter analyses of intravesicular biomarkers in the selected subpopulation. We were able to assess the total expression and phosphorylation levels of IGF-1R in non-small-cell lung cancer patients by probing plasma exosomes as a non-invasive alternative to conventional tissue biopsy. We foresee that the microfluidic exosome analysis platform will form the basis for critically needed infrastructures for advancing the biology and clinical utilization of exosomes. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Particle analysis None

	EV140164	1/1	Leishmania major	Parasite	(d)(U)C DG Filtration	Hassani K	2014	22%
Study summary Full title Absence of metalloprotease GP63 alters the protein content of Leishmania exosomes All authors Hassani K, Shio MT, Martel C, Faubert D, Olivier M Journal PLoS One Abstract Protozoan parasites of Leishmania genus are able to successfully infect their host macrophage due to (show more...)Protozoan parasites of Leishmania genus are able to successfully infect their host macrophage due to multiple virulence strategies that result in its deactivation. Recent studies suggest Leishmania GP63 to be a critical virulence factor in modulation of many macrophage molecules, including protein tyrosine phosphatases (PTPs) and transcription factors (TFs). Additionally, we and others recently reported that Leishmania-released exosomes can participate in pathogenesis. Exosomes are 40-100 nm vesicles that are freed by many eukaryotic cells. To better understand the GP63-dependent immune modulation of the macrophage by Leishmania parasites and their exosomes, we compared the immunomodulatory properties of Leishmania major (WT) and L. major gp63-/- (KO) as well as their exosomes in vitro and in vivo. Importantly, we observed that Leishmania exosomes can modulate macrophage PTPs and TFs in a GP63-dependent manner. In addition, our qRT-PCR analyses showed that WT parasites were able to downregulate multiple genes involved in the immune response, especially cytokines and pattern recognition receptors. KO parasites showed a strongly reduced modulatory capacity compared to WT parasites. Furthermore, comparison of WT versus KO exosomes also showed divergences in alteration of gene expression, especially of chemokine receptors. In parallel, studying the in vivo inflammatory recruitment using a murine air pouch model, we found that exosomes have stronger proinflammatory properties than parasites and preferentially induce the recruitment of neutrophils. Finally, comparative proteomics of WT and KO exosomes surprisingly revealed major differences in their protein content, suggesting a role for GP63 in Leishmania exosomal protein sorting. Collectively our data clearly establish the crucial role of GP63 in dampening the innate inflammatory response during early Leishmania infection, and also provides new insights in regard to the role and biology of exosomes in Leishmania host-parasite interactions. (hide) EV-METRIC 22% (30th 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 Parasite Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: PTP1B/ LACK/ PTPN6 / Tubulin/ HSP83/ GP63/ TCPTP non-EV: Proteomics yes Show all info Study aim Function Sample Species Leishmania major Sample Type Parasite Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Density gradient Highest density fraction 2 Orientation Top-down Speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins "PTPN6 / PTP1B/ TCPTP / GP63/ LACK/ HSP83/ Tubulin" ELISA Antibody details provided? No Detected EV-associated proteins "PTPN6 / PTP1B/ TCPTP / GP63/ LACK/ HSP83/ Tubulin" Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140161	1/1	Mus musculus	NAY	(d)(U)C	Grey M	2014	22%
Study summary Full title Acceleration of alpha-Synuclein Aggregation by Exosomes All authors Grey M, Dunning CJ, Gaspar R, Grey C, Brundin P, Sparr E, Linse S Journal J Biol Chem Abstract Exosomes are small vesicles released from cells into extracellular space. We have isolated exosomes (show more...)Exosomes are small vesicles released from cells into extracellular space. We have isolated exosomes from neuroblastoma cells and investigated their influence on the aggregation of ?-synuclein, a protein associated with Parkinson disease pathology. Using cryo-transmission electron microscopy of exosomes, we found spherical unilamellar vesicles with a significant protein content, and Western blot analysis revealed that they contain, as expected, the proteins Flotillin-1 and Alix. Using thioflavin T fluorescence to monitor aggregation kinetics, we found that exosomes catalyze the process in a similar manner as a low concentration of preformed ?-synuclein fibrils. The exosomes reduce the lag time indicating that they provide catalytic environments for nucleation. The catalytic effects of exosomes derived from naive cells and cells that overexpress ?-synuclein do not differ. Vesicles prepared from extracted exosome lipids accelerate aggregation, suggesting that the lipids in exosomes are sufficient for the catalytic effect to arise. Using mass spectrometry, we found several phospholipid classes in the exosomes, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and the gangliosides GM2 and GM3. Within each class, several species with different acyl chains were identified. We then prepared vesicles from corresponding pure lipids or defined mixtures, most of which were found to retard ?-synuclein aggregation. As a striking exception, vesicles containing ganglioside lipids GM1 or GM3 accelerate the process. Understanding how ?-synuclein interacts with biological membranes to promote neurological disease might lead to the identification of novel therapeutic targets. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ Flotilin1/ GAPDH 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 serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 90 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ Flotilin1/ GAPDH Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins GAPDH Characterization: Particle analysis DLS NTA

	EV140077	1/1	Mus musculus	NAY	(d)(U)C	Grad LI	2014	22%
Study summary Full title Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms All authors Grad LI, Yerbury JJ, Turner BJ, Guest WC, Pokrishevsky E, O'Neill MA, Yanai A, Silverman JM, Zeineddine R, Corcoran L, Kumita JR, Luheshi LM, Yousefi M, Coleman BM, Hill AF, Plotkin SS, Mackenzie IR, Cashman NR Journal Proc Natl Acad Sci U S A Abstract Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic (show more...)Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ Flotilin1 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ TSG101 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein SOD1;PrP Image type Close-up, Wide-field

	EV140160	1/1	Rattus norvegicus/rattus	Coronary perfusate	(d)(U)C Filtration	Giricz Z	2014	22%
Study summary Full title Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular vesicles All authors Giricz Z, Varga ZV, Baranyai T, Sipos P, Pálóczi K, Kittel Á, Buzás EI, Ferdinandy P Journal J Mol Cell Cardiol Abstract Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on (show more...)Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30-100 nm and 100-1000 nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, renders EV ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3 × 5-5 min global ischemia and reperfusion (IPC) or 30 min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30 min global ischemia and 120 min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9 ± 1.6% vs. 25.0 ± 2.7%), similarly to cardioprotection afforded by IPC (7.3 ± 2.7% vs. 22.1 ± 2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0 ± 2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection. (hide) EV-METRIC 22% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 Coronary perfusate Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 115.4 (pelleting) Protein markers EV: HSP60 non-EV: Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Coronary perfusate Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type MLA55 Pelleting: adjusted k-factor 115.4 Filtration steps > 0.45 µm, 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP60 ELISA Antibody details provided? No Detected EV-associated proteins HSP60 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV140074	1/1	Homo sapiens	NAY	(d)(U)C	Ghossoub R	2014	22%
Study summary Full title Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2 All authors Ghossoub R, Lembo F, Rubio A, Gaillard CB, Bouchet J, Vitale N, Slavík J, Machala M, Zimmermann P Journal Nat Commun Abstract Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communic (show more...)Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD63/ Syndecan1/ Syntenin non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 180 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ CD63/ Syntenin/ Syndecan1 ELISA Antibody details provided? No Detected EV-associated proteins Syndecan1 Characterization: Particle analysis None

	EV140152	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Effenberger T	2014	22%
Study summary Full title Senescence-associated release of transmembrane proteins involves proteolytic processing by ADAM17 and microvesicle shedding All authors Effenberger T, von der Heyde J, Bartsch K, Garbers C, Schulze-Osthoff K, Chalaris A, Murphy G, Rose-John S, Rabe B Journal FASEB J Abstract Cellular senescence, a state of persistent cell cycle arrest, has emerged as a potent tumor suppress (show more...)Cellular senescence, a state of persistent cell cycle arrest, has emerged as a potent tumor suppressor mechanism by restricting proliferation of cells at risk for neoplastic transformation. Senescent cells secrete various growth factors, cytokines, and other proteins that can either elicit the clearance of tumor cells or potentially promote tumor progression. In addition, this senescence-associated secretory phenotype (SASP) includes various factors that are synthesized as transmembrane precursors and subsequently converted into their soluble counterparts. Despite the importance of the SASP to tumor biology, it is virtually unknown how transmembrane proteins are released from senescent cancer cells. Here we show in different models of senescence that the metalloprotease A disintegrin and metalloproteinase 17 (ADAM17) is activated and releases the epidermal growth factor receptor ligand amphiregulin and tumor necrosis factor receptor I (TNFRI) from the surface of senescent cells by ectodomain shedding. ADAM17 activation involves phosphorylation of its cytoplasmic tail by mitogen-activated protein kinase (MAPK) p38. Interestingly, unlike amphiregulin and TNFRI, full-length intercellular adhesion molecule 1 (ICAM1) is released from senescent cells by microvesicles independently of ADAM17. Thus, our results suggest that transmembrane proteins can be released by two distinct mechanisms and point to a crucial role for ADAM17 in shaping the secretory profile of senescent cells. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 156.9 (pelleting) Protein markers EV: TSG101/ HSP70/ ICAM1/ ADAM-10 non-EV: Proteomics no Show all info Study aim Other/TM protein release from senescent cancer cells Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 150 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101/ ADAM-10/ ICAM1 ELISA Antibody details provided? No Detected EV-associated proteins ADAM-10/ ICAM1 Characterization: Particle analysis None

	EV140069	1/1	Homo sapiens	NAY	(d)(U)C	Dovrat S	2014	22%
Study summary Full title 14-3-3 and beta-catenin are secreted on extracellular vesicles to activate the oncogenic Wnt pathway All authors Dovrat S, Caspi M, Zilberberg A, Lahav L, Firsow A, Gur H, Rosin-Arbesfeld R Journal Mol Oncol Abstract Aberrant activation of the canonical Wnt signal transduction pathway is involved in a large number o (show more...)Aberrant activation of the canonical Wnt signal transduction pathway is involved in a large number of human diseases. ?-catenin, the key effector protein of the canonical Wnt pathway, functions in the nucleus with T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate expression of Wnt target genes. Here we show that members of the 14-3-3 protein family bind disheveled-2 (Dvl-2) and glycogen synthase-3? (GSK-3?) to attenuate the interaction between GSK-3? and ?-catenin. Importantly, 14-3-3 and ?-catenin form bleb-like structures and are secreted via extracellular vesicles to induce Wnt signaling activity in target cells. Our data suggest a novel way of transducing the oncogenic Wnt signal in which ?-catenin is regulated by 14-3-3? through the formation of oncosomes that contain both the 14-3-3 and ?-catenin proteins. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: HSP70/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ HSP70 Characterization: Particle analysis None

	EV140288	3/3	Mus musculus	Brain tissue	(d)(U)C DG Filtration	Dinkins MB	2014	22%
Study summary Full title Exosome reduction in vivo is associated with lower amyloid plaque load in the 5XFAD mouse model of Alzheimer's disease All authors Dinkins MB, Dasgupta S, Wang G, Zhu G, Bieberich E Journal Neurobiol Aging Abstract We present evidence here that exosomes stimulate aggregation of amyloid beta (A?)1-42 in vitro and i (show more...)We present evidence here that exosomes stimulate aggregation of amyloid beta (A?)1-42 in vitro and in vivo and interfere with uptake of A? by primary cultured astrocytes and microglia in vitro. Exosome secretion is prevented by the inhibition of neutral sphingomyelinase 2 (nSMase2), a key regulatory enzyme generating ceramide from sphingomyelin, with GW4869. Using the 5XFAD mouse, we show that intraperitoneal injection of GW4869 reduces the levels of brain and serum exosomes, brain ceramide, and A?1-42 plaque load. Reduction of total A?1-42 as well as number of plaques in brain sections was significantly greater (40% reduction) in male than female mice. Our results suggest that GW4869 reduces amyloid plaque formation in vivo by preventing exosome secretion and identifies nSMase2 as a potential drug target in AD by interfering with exosome secretion. (hide) EV-METRIC 22% (12th 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 Brain tissue Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: Alix/ TSG101 non-EV: Proteomics no EV density (g/ml) 1.18-1.27 Show all info Study aim Function Sample Species Mus musculus Sample Type Brain tissue Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 70 Wash: volume per pellet (ml) 60 Density gradient Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101 Characterization: Particle analysis None

	EV140118	1/3	Sus scrofa	NAY	(d)(U)C	Comelli L	2014	22%
Study summary Full title Characterization of secreted vesicles from vascular smooth muscle cells All authors Comelli L, Rocchiccioli S, Smirni S, Salvetti A, Signore G, Citti L, Trivella MG, Cecchettini A Journal Mol Biosyst Abstract The artery medial layer is mainly composed of vascular smooth muscle cells (VSMCs). These cells cont (show more...)The artery medial layer is mainly composed of vascular smooth muscle cells (VSMCs). These cells contribute to the formation of neointima and atherosclerotic plaques by switching from the quiescent-contractile to migratory-activated state. Apoptotic blebs, microvesicles and exosomes are secreted vesicles, with differences in composition and size, involved in cellular communication at multiple levels. In this article, an untargeted, proteomics approach was exploited to characterise VSMC released vesicles and a preliminary protein profile for microvesicles and exosomes of different cell phenotypes was obtained. Enriched samples of vesicles from serum-free and serum-activated VSMCs were analysed by a LC-MS/MS strategy leading to the identification of 349 proteins. In microvesicles, the most abundant classes of identified proteins were cytoplasmic or organelle associated, house keeping and metabolic factors. Otherwise, exosomes from different phenotypes revealed a sharper peculiarity thus, as suggested by the high percentage of ECM and ECM related proteins and cell adhesion molecules, they seem to play an important role in outward or cell-to-cell signalling. Comparison between exosomes or microvesicles from quiescent and activated VSMCs evidenced 29 differentially expressed proteins. Among these, in microvesicles there are several proteins that are involved in vesicle trafficking while in exosomes focal adhesion and ECM related factors are the most interesting. These data, although preliminary, are promising for a possible identification of potential circulating markers of a cell state. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Prohibitin non-EV: Proteomics yes Show all info Study aim Omics Sample Species Sus scrofa Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 30 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Prohibitin ELISA Antibody details provided? No Detected EV-associated proteins Prohibitin Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV140118	2/3	Sus scrofa	NAY	(d)(U)C	Comelli L	2014	22%
Study summary Full title Characterization of secreted vesicles from vascular smooth muscle cells All authors Comelli L, Rocchiccioli S, Smirni S, Salvetti A, Signore G, Citti L, Trivella MG, Cecchettini A Journal Mol Biosyst Abstract The artery medial layer is mainly composed of vascular smooth muscle cells (VSMCs). These cells cont (show more...)The artery medial layer is mainly composed of vascular smooth muscle cells (VSMCs). These cells contribute to the formation of neointima and atherosclerotic plaques by switching from the quiescent-contractile to migratory-activated state. Apoptotic blebs, microvesicles and exosomes are secreted vesicles, with differences in composition and size, involved in cellular communication at multiple levels. In this article, an untargeted, proteomics approach was exploited to characterise VSMC released vesicles and a preliminary protein profile for microvesicles and exosomes of different cell phenotypes was obtained. Enriched samples of vesicles from serum-free and serum-activated VSMCs were analysed by a LC-MS/MS strategy leading to the identification of 349 proteins. In microvesicles, the most abundant classes of identified proteins were cytoplasmic or organelle associated, house keeping and metabolic factors. Otherwise, exosomes from different phenotypes revealed a sharper peculiarity thus, as suggested by the high percentage of ECM and ECM related proteins and cell adhesion molecules, they seem to play an important role in outward or cell-to-cell signalling. Comparison between exosomes or microvesicles from quiescent and activated VSMCs evidenced 29 differentially expressed proteins. Among these, in microvesicles there are several proteins that are involved in vesicle trafficking while in exosomes focal adhesion and ECM related factors are the most interesting. These data, although preliminary, are promising for a possible identification of potential circulating markers of a cell state. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles Vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ TSG101/ Prohibitin non-EV: Proteomics yes Show all info Study aim Omics Sample Species Sus scrofa Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101/ Prohibitin ELISA Antibody details provided? No Detected EV-associated proteins Prohibitin Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV140147	3/3	Homo sapiens	NAY	(d)(U)C Filtration	Cheng L	2014	22%
Study summary Full title Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine All authors Cheng L, Sun X, Scicluna BJ, Coleman BM, Hill AF Journal Kidney Int Abstract Micro RNAs (miRNAs) have been shown to circulate in biological fluids and are enclosed in vesicles s (show more...)Micro RNAs (miRNAs) have been shown to circulate in biological fluids and are enclosed in vesicles such as exosomes; they are present in urine and represent a noninvasive methodology to detect biomarkers for diagnostic testing. The low abundance of RNA in urine creates difficulties in its isolation, of which exosomal miRNA is a small fraction, making downstream RNA assays challenging. Here, we investigate methods to maximize exosomal isolation and RNA yield for next-generation deep sequencing. Upon characterizing exosomal proteins and total RNA content in urine, several commercially available kits were tested for their RNA extraction efficiency. We subsequently used the methods with the highest miRNA content to profile baseline miRNA expression using next-generation deep sequencing. Comparisons of miRNA profiles were also made with exosomes isolated by differential ultracentrifugation methodology and a commercially available column-based protocol. Overall, miRNAs were found to be significantly enriched and intact in urine-derived exosomes compared with cell-free urine. The presence of other noncoding RNAs such as small nuclear and small nucleolar RNA in the exosomes, in addition to coding sequences related to kidney and bladder conditions, was also detected. Our study extensively characterizes the RNA content of exosomes isolated from urine, providing the potential to identify miRNA biomarkers in human urine. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ Flotillin non-EV: Cell organelle protein Proteomics no Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ Flotillin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Flotillin Characterization: Particle analysis None

	EV140146	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Chen WX	2014	22%
Study summary Full title Exosomes from drug-resistant breast cancer cells transmit chemoresistance by a horizontal transfer of microRNAs All authors Chen WX, Liu XM, Lv MM, Chen L, Zhao JH, Zhong SL, Ji MH, Hu Q, Luo Z, Wu JZ, Tang JH Journal PLoS One Abstract Adriamycin and docetaxel are two agents commonly used in treatment of breast cancer, but their effic (show more...)Adriamycin and docetaxel are two agents commonly used in treatment of breast cancer, but their efficacy is often limited by the emergence of chemoresistance. Recent studies indicate that exosomes act as vehicles for exchange of genetic cargo between heterogeneous populations of tumor cells, engendering a transmitted drug resistance for cancer development and progression. However, the specific contribution of breast cancer-derived exosomes is poorly understood. Here we reinforced other's report that human breast cancer cell line MCF-7/S could acquire increased survival potential from its resistant variants MCF-7/Adr and MCF-7/Doc. Additionally, exosomes of the latter, A/exo and D/exo, significantly modulated the cell cycle distribution and drug-induced apoptosis with respect to S/exo. Exosomes pre-treated with RNase were unable to regulate cell cycle and apoptosis resistance, suggesting an RNA-dependent manner. Microarray and polymerase chain reaction for the miRNA expression profiles of A/exo, D/exo, and S/exo demonstrated that they loaded selective miRNA patterns. Following A/exo and D/exo transfer to recipient MCF-7/S, the same miRNAs were significantly increased in acquired cells. Target gene prediction and pathway analysis showed the involvement of miR-100, miR-222, and miR-30a in pathways implicated in cancer pathogenesis, membrane vesiculation and therapy failure. Furthermore, D/exo co-culture assays and miRNA mimics transfection experiments indicated that miR-222-rich D/exo could alter target gene expression in MCF-7/S. Our results suggest that drug-resistant breast cancer cells may spread resistance capacity to sensitive ones by releasing exosomes and that such effects could be partly attributed to the intercellular transfer of specific miRNAs. (hide) EV-METRIC 22% (59th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ CD44/ Beta-actin non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ Beta-actin/ CD44 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin/ CD44 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140064	1/2	Homo sapiens	Serum	(d)(U)C DC	Caradec J	2014	22%
Study summary Full title Reproducibility and efficiency of serum-derived exosome extraction methods All authors Caradec J, Kharmate G, Hosseini-Beheshti E, Adomat H, Gleave M, Guns E Journal Clin Biochem Abstract OBJECTIVES: Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic (show more...)OBJECTIVES: Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum. DESIGN AND METHODS: We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94. RESULTS: Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG. CONCLUSION: ExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes. (hide) EV-METRIC 22% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation 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 NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Protein markers EV: LAMP2/ CD9 non-EV: Albumin/ HSP90B1/ Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ LAMP2 Detected contaminants Cell organelle protein/ Albumin/ HSP90B1 ELISA Antibody details provided? No Detected EV-associated proteins LAMP2 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

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