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Results list Most used isolation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV130025	2/2	Salmonella enterica	Bacteria	0.2 µm filter dUC Sucrose-DG (valid.)	Guidi R	2013	78%
Study summary Full title Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells All authors Guidi R, Levi L, Rouf SF, Puiac S, Rhen M, Frisan T Journal Cell Microbiol Abstract Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are pro (show more...)Cytolethal-distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram-negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella-containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71-CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71-CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71-CDT also secreted OMVs-like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin-loaded OMVs by bystander cells was dependent on dynamin-1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria. (hide) EV-METRIC 78% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bacteria Focus vesicles OMV Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Adj. k-factor 602 (pelleting) / 602 (washing) Protein markers EV: None non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.130 TEM measurements 50 Show all info Study aim Function Sample Species Salmonella enterica Sample Type Bacteria Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 70 Pelleting: rotor type TH641 Pelleting: adjusted k-factor 602.0 Wash: Rotor Type TH641 Wash: adjusted k-factor 602.0 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.2 Highest density fraction 2 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV130022	1/3	Homo sapiens Mus musculus	Cell culture supernatant	dUC Sucrose-DG (valid.)	Zhu H	2013	78%
Study summary Full title Mutation of SIMPLE in Charcot-Marie-Tooth 1C alters production of exosomes All authors Zhu H, Guariglia S, Yu RY, Li W, Brancho D, Peinado H, Lyden D, Salzer J, Bennett C, Chow CW Journal Mol Biol Cell Abstract Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small inte (show more...)Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis. (hide) EV-METRIC 78% (98th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ CD63/ Flotilin1/ HSP70/ Hsc70/ SIMPLE non-EV: Tubulin Proteomics no EV density (g/ml) 1.130 TEM measurements 40-100 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Top-down Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ Flotilin1/ HSP70/ Hsc70/ SIMPLE Detected contaminants Tubulin ELISA Detected EV-associated proteins Hsc70/ SIMPLE Characterization: Particle analysis NTA EM EM-type immune EM/ scanning EM Image type Close-up, Wide-field

	EV130001	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	Yoshioka Y	2013	67%
Study summary Full title Comparative marker analysis of extracellular vesicles in different human cancer types All authors Yoshioka Y, Konishi Y, Kosaka N, Katsuda T, Kato T, Ochiya T Journal J Extracell Vesicles Abstract Several cell types, including tumour cells, secrete extracellular vesicles (EVs), and tumour-derived (show more...)Several cell types, including tumour cells, secrete extracellular vesicles (EVs), and tumour-derived EVs play a role in cancer initiation and progression. These vesicles include both a common set of membrane and cytosolic proteins and origin-specific subsets of proteins that likely correlated to cell type-associated functions. To confirm the presence of EVs in the preparations, researchers have identified so-called EV marker proteins, including the tetraspanin family proteins and such cytosolic proteins as heat shock 70 kDa protein 4 (HSP70) and tumour susceptibility gene 101 (TSG101). However, studies have shown that some EV markers are not always present in all EVs, which not only complicates the identification of EVs but also precludes the quantitative evaluation of EV proteins. Thus, it is strongly required to explore well-conserved EV marker proteins that are present at similar levels, regardless of their tissue or cellular origin. In this study, we compared the presence of 11 well-known EV marker proteins by immunoblotting using EVs isolated from 4 human prostate cell lines and 5 human breast cell lines, including cancer cells with different phenotypes. We found that all the tested EVs were positive for CD9 and CD81, with similar abundance that was irrespective of the EV origin. In contrast, other EV marker proteins, such as TSG101, Rab-5b and CD63, were detected in an inconsistent manner, depending on the origin of the EVs. Thus, we propose that the detection of CD9 and/or CD81 should ensure the presence of EVs. (hide) EV-METRIC 67% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Protein markers EV: CD63/ CD81/ CD9/ Flotilin1/ HSP70/ TSG101/ Caveolin1/ Rab5b/ Annexin2/ Integrin-beta1/ Actin non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Wash: volume per pellet (ml) 11 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ CD9/ Flotilin1/ HSP70/ TSG101/ Caveolin1/ Rab5b/ Annexin2/ Integrin-beta1/ Actin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Caveolin1/ Rab5b/ Annexin2/ Integrin-beta1/ Actin Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV130002	1/2	Homo sapiens	Cell culture supernatant	Density cushion dUC Sucrose-DG (valid.)	Liang B	2013	67%
Study summary Full title Characterization and proteomic analysis of ovarian cancer-derived exosomes All authors Liang B, Peng P, Chen S, Li L, Zhang M, Cao D, Yang J, Li H, Gui T, Li X, Shen K Journal J Proteomics Abstract Ovarian cancer is the most lethal type of cancer among all frequent gynecologic malignancies, becaus (show more...)Ovarian cancer is the most lethal type of cancer among all frequent gynecologic malignancies, because most patients present with advanced disease at diagnosis. Exosomes are important intercellular communication vehicles, released by various cell types. Here we presented firstly the protein profile of highly purified exosomes derived from two ovarian cancer cell lines, OVCAR-3 and IGROV1. The exosomes derived from ovarian cancer cell lines were round and mostly 30-100 nm in diameter when viewed under an electron microscope. The exosomal marker proteins TSG101 and Alix were detected in exosome preparations. The range of density was between 1.09 g/ml and 1.15 g/ml. A total of 2230 proteins were identified from two ovarian cell-derived exosomes. Among them, 1017 proteins were identified in both exosomes including all of the major exosomal protein markers. There were 380 proteins that are not reported in the ExoCarta database. In addition to common proteins from exosomes of various origins, our results showed that ovarian cancer-derived exosomes also carried tissue specific proteins associated with tumorigenesis and metastasis, especially in ovarian carcinoma. Based on the known roles of exosomes in cellular communication, these data indicate that exosomes released by ovarian cancer cells may play important roles in ovarian cancer progression and provide a potential source of blood-based protein biomarkers. (hide) EV-METRIC 67% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Density cushion + dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ TSG101/ EpCAM/ Actin non-EV: Cell organelle protein/ hnRNPA2/ hnRNPK Proteomics yes EV density (g/ml) 1.1-1.14 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 80 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 120000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ TSG101/ EpCAM/ Actin Detected contaminants Cell organelle protein/ hnRNPA2/ hnRNPK ELISA Detected EV-associated proteins EpCAM/ Actin Characterization: Particle analysis EM EM-type cryo EM Image type Close-up, Wide-field

	EV130018	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC Sucrose-DG (valid.)	Skogberg G	2013	63%
Study summary Full title Characterization of human thymic exosomes All authors Skogberg G, Gudmundsdottir J, van der Post S, Sandström K, Bruhn S, Benson M, Mincheva-Nilsson L, Baranov V, Telemo E, Ekwall O Journal PLoS One Abstract Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capab (show more...)Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla. (hide) EV-METRIC 63% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Protein markers EV: CD63/ CD81/ CD9/ TSG101/ MHC2/ MFGE8/ ICAM1 non-EV: "TGF-beta/ CD3/ CD4/ CD8/ EpCAM" Proteomics yes EV density (g/ml) 1.15-1.19 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Density gradient Only used for validation of main results 1 Density medium Sucrose Orientation Top-down Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins MHC2/ MFGE8/ ICAM1 ELISA Detected EV-associated proteins MHC2/ MFGE8/ ICAM1 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV130047	1/1	Mus musculus	Cell culture supernatant	0.2 µm filter dUC Sucrose-DG (valid.)	Näslund TI	2013	57%
Study summary Full title Dendritic cell-derived exosomes need to activate both T and B cells to induce antitumor immunity All authors Näslund TI, Gehrmann U, Qazi KR, Karlsson MC, Gabrielsson S Journal J Immunol Abstract Exosomes are secreted membrane nanovesicles of endosomal origin and are considered potential cancer (show more...)Exosomes are secreted membrane nanovesicles of endosomal origin and are considered potential cancer vaccine vectors. Phase I clinical trials have been successfully conducted with tumor peptide-loaded exosomes derived from dendritic cells (dexosomes), and a phase II clinical trial is ongoing. However, much is still unknown regarding the in vivo role of dexosomes and whether their immunogenicity can be enhanced. We previously reported that dexosomes induce CD4(+) T cell responses in a B cell-dependent manner, suggesting that immunization with dexosomes carrying only T cell peptides induce suboptimal immune responses. In this study, we show that CD8(+) T cell responses were induced in vivo when mice were immunized with protein-loaded, but not peptide-loaded, dexosomes. We also show that the cytotoxic T cell response was totally dependent on CD4(+) T cells and, interestingly, also on B cells. Mice deficient in complement activation and Ag shuttling by B cells have lower responses to protein-loaded dexosomes, showing involvement of these B cell-mediated mechanisms. Finally, protein-loaded dexosomes were superior in protecting against tumor growth. In conclusion, proper activation of CD4(+) T and B cells needs to be considered when designing cancer vaccines to ensure full potential of the treatment. (hide) EV-METRIC 57% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Protein markers EV: CD81/ CD9/ MHC2 non-EV: CD54/ CD80/ CD86 Proteomics no EV density (g/ml) 1.11-1.19 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 130 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 80000 Filtration steps 0.22µm or 0.2µm Western Blot Detected EV-associated proteins MHC2 ELISA Detected EV-associated proteins MHC2 Flow cytometry specific beads Selected surface protein(s) Yes Characterization: Particle analysis DLS Particle analysis: flow cytometry

	EV130022	2/3	Homo sapiens	CMT1C patient B cells	dUC Sucrose-DG (valid.)	Zhu H	2013	56%
Study summary Full title Mutation of SIMPLE in Charcot-Marie-Tooth 1C alters production of exosomes All authors Zhu H, Guariglia S, Yu RY, Li W, Brancho D, Peinado H, Lyden D, Salzer J, Bennett C, Chow CW Journal Mol Biol Cell Abstract Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small inte (show more...)Charcot-Marie-Tooth (CMT) disease is an inherited neurological disorder. Mutations in the small integral membrane protein of the lysosome/late endosome (SIMPLE) account for the rare autosomal-dominant demyelination in CMT1C patients. Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by perplexity about the role of SIMPLE, which is expressed in multiple cell types. Here we show that SIMPLE resides within the intraluminal vesicles of multivesicular bodies (MVBs) and inside exosomes, which are nanovesicles secreted extracellularly. Targeting of SIMPLE to exosomes is modulated by positive and negative regulatory motifs. We also find that expression of SIMPLE increases the number of exosomes and secretion of exosome proteins. We engineer a point mutation on the SIMPLE allele and generate a physiological mouse model that expresses CMT1C-mutated SIMPLE at the endogenous level. We find that CMT1C mouse primary embryonic fibroblasts show decreased number of exosomes and reduced secretion of exosome proteins, in part due to improper formation of MVBs. CMT1C patient B cells and CMT1C mouse primary Schwann cells show similar defects. Together the data indicate that SIMPLE regulates the production of exosomes by modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome-mediated intercellular communications may place an overwhelming burden on the nervous system and account for CMT1C molecular pathogenesis. (hide) EV-METRIC 56% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type CMT1C patient B cells Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ CD63/ "SIMPLE/ Clathrin HC" non-EV: Tubulin Proteomics no TEM measurements >100 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type CMT1C patient B cells Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ "SIMPLE/ Clathrin HC" Detected contaminants Tubulin ELISA Detected EV-associated proteins "SIMPLE/ Clathrin HC" Characterization: Particle analysis NTA EM EM-type scanning EM Image type Wide-field

	EV130058	1/1	Trichomonas vaginalis	Parasite	0.2 µm filter dUC Sucrose-DG UF	Twu O	2013	56%
Study summary Full title Trichomonas vaginalis exosomes deliver cargo to host cells and mediate hostratioparasite interactions All authors Twu O, de Miguel N, Lustig G, Stevens GC, Vashisht AA, Wohlschlegel JA, Johnson PJ Journal PLoS Pathog Abstract Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential (show more...)Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host?parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite?parasite communication and host cell colonization. (hide) EV-METRIC 56% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Parasite Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG + UF Adj. k-factor 25.32 (pelleting) Protein markers EV: Tspan1 non-EV: None Proteomics yes EV density (g/ml) 1.03-1.25 Show all info Study aim Function Sample Species Trichomonas vaginalis Sample Type Parasite Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting: time(min) 75 Pelleting: rotor type TLA100 Pelleting: adjusted k-factor 25.32 Wash: volume per pellet (ml) 2 Density gradient Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 3 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Tspan1 ELISA Detected EV-associated proteins Tspan1 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV130017	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC Sucrose-DG	Salomon C	2013	56%
Study summary Full title Hypoxia-induced changes in the bioactivity of cytotrophoblast-derived exosomes All authors Salomon C, Kobayashi M, Ashman K, Sobrevia L, Mitchell MD, Rice GE Journal PLoS One Abstract Migration of extravillous trophoblasts (EVT) into decidua and myometrium is a critical process in th (show more...)Migration of extravillous trophoblasts (EVT) into decidua and myometrium is a critical process in the conversion of maternal spiral arterioles and establishing placenta perfusion. EVT migration is affected by cell-to-cell communication and oxygen tension. While the release of exosomes from placental cells has been identified as a significant pathway in materno-fetal communication, the role of placental-derived exosomes in placentation has yet to be established. The aim of this study was to establish the effect of oxygen tension on the release and bioactivity of cytotrophoblast (CT)-derived exosomes on EVT invasion and proliferation. CT were isolated from first trimester fetal tissue (n = 12) using a trypsin-deoxyribonuclease-dispase/Percoll method. CT were cultured under 8%, 3% or 1% O2 for 48 h. Exosomes from CT-conditioned media were isolated by differential and buoyant density centrifugation. The effect of oxygen tension on exosome release (µg exosomal protein/10(6)cells/48 h) and bioactivity were established. HTR-8/SVneo (EVT) were used as target cells to establish the effect (bioactivity) of exosomes on invasion and proliferation as assessed by real-time, live-cell imaging (Incucyte™). The release and bioactivity of CT-derived exosomes were inversely correlated with oxygen tension (p<0.001). Under low oxygen tensions (i.e. 1% O2), CT-derived exosomes promoted EVT invasion and proliferation. Proteomic analysis of exosomes identified oxygen-dependent changes in protein content. We propose that in response to changes in oxygen tension, CTs modify the bioactivity of exosomes, thereby, regulating EVT phenotype. Exosomal induction of EVT migration may represent a normal process of placentation and/or an adaptive response to placental hypoxia. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG Adj. k-factor 266.3 (pelleting) Protein markers EV: CD63/ CD81/ CD9/ PLAP non-EV: None Proteomics yes EV density (g/ml) 1.15-1.2 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 75 Pelleting: rotor type SureSpin630/36 Pelleting: adjusted k-factor 266.3 Density gradient Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ CD9/ PLAP ELISA Detected EV-associated proteins PLAP Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV130024	2/2	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC Iodixanol-DG (valid.)	Narayanan A	2013	56%
Study summary Full title Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA All authors Narayanan A, Iordanskiy S, Das R, Van Duyne R, Santos S, Jaworski E, Guendel I, Sampey G, Dalby E, Iglesias-Ussel M, Popratiloff A, Hakami R, Kehn-Hall K, Young M, Subra C, Gilbert C, Bailey C, Romerio F, Kashanchi F Journal J Biol Chem Abstract Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from (show more...)Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Iodixanol-DG (valid.) Protein markers EV: Alix/ CD63/ HSP70/ AChE/ Beta-actin non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.075 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 6 Highest density fraction 18 Orientation Top-down Rotor type 70Ti Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ HSP70/ AChE/ Beta-actin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins AChE/ Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV130044	1/1	Rattus norvegicus/rattus	Cell culture supernatant	dUC Sucrose-DG (valid.)	Lopez-Verrilli MA	2013	56%
Study summary Full title Schwann cell-derived exosomes enhance axonal regeneration in the peripheral nervous system All authors Lopez-Verrilli MA, Picou F, Court FA Journal Glia Abstract Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). Af (show more...)Axonal regeneration in the peripheral nervous system is greatly supported by Schwann cells (SCs). After nerve injury, SCs dedifferentiate to a progenitor-like state and efficiently guide axons to their original target tissues. Contact and soluble factors participate in the crosstalk between SCs and axons during axonal regeneration. Here we show that dedifferentiated SCs secrete nano-vesicles known as exosomes which are specifically internalized by axons. Surprisingly, SC-derived exosomes markedly increase axonal regeneration in vitro and enhance regeneration after sciatic nerve injury in vivo. Exosomes shift the growth cone morphology to a pro-regenerating phenotype and decrease the activity of the GTPase RhoA, involved in growth cone collapse and axon retraction. Altogether, our work identifies a novel mechanism by which SCs communicate with neighboring axons during regenerative processes. We propose that SC exosomes represent an important mechanism by which these cells locally support axonal maintenance and regeneration after nerve damage. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Adj. k-factor 157.9 (pelleting) Protein markers EV: CD63/ Flotilin1/ HSP90/ HSP70/ TSG101 non-EV: Rab5 Proteomics no EV density (g/ml) 1.12-1.14 Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type T865 Pelleting: adjusted k-factor 157.9 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Flotilin1/ HSP90/ HSP70/ TSG101 Detected contaminants Rab5 Flow cytometry specific beads Selected surface protein(s) Yes Characterization: Particle analysis Particle analysis: flow cytometry EM EM-type immune EM Image type Close-up

	EV130002	2/2	Homo sapiens	Cell culture supernatant	Density cushion dUC Sucrose-DG (valid.)	Liang B	2013	56%
Study summary Full title Characterization and proteomic analysis of ovarian cancer-derived exosomes All authors Liang B, Peng P, Chen S, Li L, Zhang M, Cao D, Yang J, Li H, Gui T, Li X, Shen K Journal J Proteomics Abstract Ovarian cancer is the most lethal type of cancer among all frequent gynecologic malignancies, becaus (show more...)Ovarian cancer is the most lethal type of cancer among all frequent gynecologic malignancies, because most patients present with advanced disease at diagnosis. Exosomes are important intercellular communication vehicles, released by various cell types. Here we presented firstly the protein profile of highly purified exosomes derived from two ovarian cancer cell lines, OVCAR-3 and IGROV1. The exosomes derived from ovarian cancer cell lines were round and mostly 30-100 nm in diameter when viewed under an electron microscope. The exosomal marker proteins TSG101 and Alix were detected in exosome preparations. The range of density was between 1.09 g/ml and 1.15 g/ml. A total of 2230 proteins were identified from two ovarian cell-derived exosomes. Among them, 1017 proteins were identified in both exosomes including all of the major exosomal protein markers. There were 380 proteins that are not reported in the ExoCarta database. In addition to common proteins from exosomes of various origins, our results showed that ovarian cancer-derived exosomes also carried tissue specific proteins associated with tumorigenesis and metastasis, especially in ovarian carcinoma. Based on the known roles of exosomes in cellular communication, these data indicate that exosomes released by ovarian cancer cells may play important roles in ovarian cancer progression and provide a potential source of blood-based protein biomarkers. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Density cushion + dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ TSG101/ EpCAM/ actin non-EV: Cell organelle protein/ hnRNPA2/ hnRNPK Proteomics yes EV density (g/ml) 1.1-1.14 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 80 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 120000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ TSG101/ EpCAM/ actin Detected contaminants Cell organelle protein/ hnRNPA2/ hnRNPK ELISA Detected EV-associated proteins EpCAM/ actin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV130042	1/1	Mus musculus	Cell culture supernatant	0.2 µm filter dUC Sucrose-DG (valid.)	Kotzerke K	2013	56%
Study summary Full title Immunostimulatory activity of murine keratinocyte-derived exosomes All authors Kotzerke K, Mempel M, Aung T, Wulf GG, Urlaub H, Wenzel D, Schön MP, Braun A Journal Exp Dermatol Abstract It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble (show more...)It has long been known that keratinocytes influence cutaneous immunity through secretion of soluble factors. Exosomes, small membrane vesicles of endocytotic origin, have been implicated in intercellular communication processes such as the transfer of tumor cell antigens and the activation of recipient dendritic cells (DC). However, little is known about immunomodulatory functions of keratinocyte-derived exosomes. To address this question, we analysed exosome secretion of the murine keratinocyte cell line MPEK under steady state as well as inflammatory conditions (+/- IFN?). These exosomes were readily taken up by bone marrow-derived DC (BMDC) in vitro resulting in a matured phenotype, as evidenced by increased CD40 expression as well as by the production of large amounts of IL-6, IL-10 and IL-12. When the transfer of antigen-specific information through exosomes was investigated, it was found that keratinocytes took up antigen (ovalbumin) and transferred it to their exosomes. However, these antigen-harbouring exosomes failed to induce antigen-specific T cell responses via BMDC. Together, this novel biological function suggests that keratinocytes are able to direct unspecific immune processes but do not elicit specific immune responses. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Adj. k-factor 256 (pelleting) Protein markers EV: Alix/ Flotillin non-EV: Cell organelle protein Proteomics yes EV density (g/ml) 1.100 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 150 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.25 Orientation Top-down Rotor type SW32 Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ Flotillin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Flotillin Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV130073	5/5	Homo sapiens	Blood plasma	0.1 µm filter dUC Iodixanol-DG	Kalra H	2013	56%
Study summary Full title Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma All authors Kalra H, Adda CG, Liem M, Ang CS, Mechler A, Simpson RJ, Hulett MD, Mathivanan S Journal Proteomics Abstract Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including b (show more...)Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active. (hide) EV-METRIC 56% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.1 µm filter + dUC + Iodixanol-DG Protein markers EV: Alix/ CD63/ CD9/ Flotilin1/ HSP70/ TSG101/ TFRC non-EV: None Proteomics yes EV density (g/ml) 1.12-1.24 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Density gradient Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 1.5 Filtration steps 0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ CD9/ Flotilin1/ HSP70/ TSG101/ TFRC ELISA Detected EV-associated proteins TFRC Characterization: Particle analysis EM EM-type transmission EM/ atomic force EM Image type Close-up, Wide-field

	EV130005	1/1	Homo sapiens	Cell culture supernatant	dUC	Honegger A	2013	56%
Study summary Full title Silencing of human papillomavirus (HPV) E6/E7 oncogene expression affects both the contents and the amounts of extracellular microvesicles released from HPV-positive cancer cells All authors Honegger A, Leitz J, Bulkescher J, Hoppe-Seyler K, Hoppe-Seyler F Journal Int J Cancer Abstract The human papillomavirus (HPV) E6/E7 oncogenes play a crucial role in the HPV-induced carcinogenesis (show more...)The human papillomavirus (HPV) E6/E7 oncogenes play a crucial role in the HPV-induced carcinogenesis. In this study, the authors investigated whether silencing of endogenous HPV E6/E7 expression may influence the contents or amounts of extracellular microvesicles (eMVs) released from HPV-positive cancer cells. It was found that eMVs secreted from HeLa cells are enriched for Survivin protein. RNA interference studies revealed that maintenance of both intracellular and microvesicular Survivin amounts was strongly dependent on continuous E6/E7 expression. This indicates that intracellular HPV activities are translated into visible alterations of protein contents in eMVs. Besides Survivin, eMVs from HeLa cells contain additional members of the inhibitor of apoptosis protein (IAP) family (XIAP, c-IAP1 and Livin). In contrast, no evidence for the presence of the HPV E6 and E7 oncoproteins in eMVs was obtained. Moreover, it was found that silencing of HPV E6/E7 expression led to a significant increase of exosomes-representing eMVs of endocytic origin-released from HeLa cells. This effect was associated with the reinduction of p53, stimulation of the p53 target genes TSAP6 and CHMP4C that can enhance exosome production and induction of senescence. Taken together, these results show that silencing of HPV E6/E7 oncogene expression profoundly affects both the composition and amounts of eMVs secreted by HPV-positive cancer cells. This indicates that HPVs can induce molecular signatures in eMVs that may affect intercellular communication and could be explored for diagnostic purposes. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles microvesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 253.9 (pelleting) / 253.9 (washing) Protein markers EV: CD63/ CD9/ TSG101/ Beta-actin/ Annexin1/ Hsc70/ Survivin non-EV: Cell organelle protein Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Wash: volume per pellet (ml) 36 Wash: Rotor Type SW28 Wash: adjusted k-factor 253.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD9/ TSG101/ Beta-actin/ Annexin1/ Hsc70/ Survivin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Beta-actin/ Annexin1/ Hsc70/ Survivin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV130009	1/1	Homo sapiens	Cell culture supernatant	Density cushion (valid.) dUC Microfluidics Sucrose-DG (valid.)	Frühbeis C	2013	56%
Study summary Full title Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication All authors Frühbeis C, Fröhlich D, Kuo WP, Amphornrat J, Thilemann S, Saab AS, Kirchhoff F, Möbius W, Goebbels S, Nave KA, Schneider A, Simons M, Klugmann M, Trotter J, Krämer-Albers EM Journal PLoS Biol Abstract Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, b (show more...)Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²? entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Density cushion (valid.) + dUC + Microfluidics + Sucrose-DG (valid.) Adj. k-factor 276.6 (pelleting) Protein markers EV: Alix/ CD9/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.120 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type SW40 Pelleting: adjusted k-factor 276.6 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.3 Highest density fraction 1.8 Orientation Top-down Other Name other isolation method Microfluidics Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD9/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV130023	2/2	Drosophila melanogaster	Drosophila	dUC Sucrose-DG (valid.)	Beckett K	2013	56%
Study summary Full title Drosophila S2 cells secrete wingless on exosome-like vesicles but the wingless gradient forms independently of exosomes All authors Beckett K, Monier S, Palmer L, Alexandre C, Green H, Bonneil E, Raposo G, Thibault P, Le Borgne R, Vincent JP Journal Traffic Abstract Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls gr (show more...)Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless-expressing Drosophila S2 cells, Wingless is present on exosome-like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless-containing exosome-like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome-like vesicles. Using these exosome markers and a cell-based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless-expressing cells. (hide) EV-METRIC 56% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Drosophila Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: Wg non-EV: Cell organelle protein Proteomics yes EV density (g/ml) 1.14-1.19 Show all info Study aim Function Sample Species Drosophila melanogaster Sample Type Drosophila Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Wg Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Wg Characterization: Particle analysis EM EM-type immune EM Image type Wide-field

	EV130021	1/1	Canis familiaris	Cell culture supernatant	0.1 µm filter Iodixanol-DG UF	Tauro BJ	2013	50%
Study summary Full title Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition All authors Tauro BJ, Mathias RA, Greening DW, Gopal SK, Ji H, Kapp EA, Coleman BM, Hill AF, Kusebauch U, Hallows JL, Shteynberg D, Moritz RL, Zhu HJ, Simpson RJ Journal Mol Cell Proteomics Abstract Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the los (show more...)Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells. (hide) EV-METRIC 50% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.1 µm filter + Iodixanol-DG + UF Protein markers EV: Alix/ TSG101/ EpCAM non-EV: None Proteomics yes EV density (g/ml) 1.090 Show all info Study aim Function Sample Species Canis familiaris Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Density gradient Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 1 Filtration steps 0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ TSG101/ EpCAM ELISA Detected EV-associated proteins EpCAM Characterization: Particle analysis EM EM-type cryo EM Image type Wide-field

	EV130003	1/1	Homo sapiens	Cell culture supernatant	Commercial	Camacho L	2013	50%
Study summary Full title MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes All authors Camacho L, Guerrero P, Marchetti D Journal PLoS One Abstract Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercel (show more...)Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM) versus non-brain metastatic (non-BM) cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM) variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210) and two down-regulated miRNAs (miR-19a and miR-29c) in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis. (hide) EV-METRIC 50% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Commercial Adj. k-factor 79.91 (pelleting) Protein markers EV: CD63/ CD81/ CD9 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting: adjusted k-factor 79.91 Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ CD9 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV130137	3/3	Mus musculus Rattus norvegicus/rattus	Cell culture supernatant	0.2 µm filter Density cushion (valid.) dUC Sucrose-DG (valid.)	Royo F	2013	44%
Study summary Full title Transcriptome of extracellular vesicles released by hepatocytes All authors Royo F, Schlangen K, Palomo L, Gonzalez E, Conde-Vancells J, Berisa A, Aransay AM, Falcon-Perez JM Journal PLoS One Abstract The discovery that the cells communicate through emission of vesicles has opened new opportunities f (show more...)The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Density cushion (valid.) + dUC + Sucrose-DG (valid.) Protein markers EV: CD81/ Flotilin1/ TSG101/ Aip1 non-EV: None Proteomics no EV density (g/ml) 1.12-1.2;1.19-1.23 Show all info Study aim Omics Sample Species Mus musculus / Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Flotilin1/ TSG101/ Aip1 ELISA Detected EV-associated proteins Aip1 Characterization: Particle analysis NTA EM EM-type cryo EM Image type Close-up

	EV130016	1/1	Homo sapiens	Urine	dUC Iodixanol-DG (valid.)	Raimondo F	2013	44%
Study summary Full title Differential protein profiling of renal cell carcinoma urinary exosomes All authors Raimondo F, Morosi L, Corbetta S, Chinello C, Brambilla P, Della Mina P, Villa A, Albo G, Battaglia C, Bosari S, Magni F, Pitto M Journal Mol Biosyst Abstract Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is incr (show more...)Renal cell carcinoma (RCC) accounts for about 3% of all human malignancies and its incidence is increasing. There are no standard biomarkers currently used in the clinical management of patients with renal cell carcinoma. A promising strategy for new biomarker detection is comparative proteomics of urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, enriched in renal proteins and excluding high-abundance plasmatic proteins, such as albumin. Aim of the work is to establish the protein profile of exosomes isolated from urines of RCC patient compared with control subjects. We enrolled 29 clear cell RCC patients and 23 control healthy subjects (CTRL), age and sex-matched, for urine collection and vesicle isolation by differential centrifugation. Such vesicles were morphologically and biochemically characterized and proved to share exosome properties. Proteomic analysis, performed on 9 urinary exosome (UE) pooled samples by gel based digestion followed by LC-MS/MS, led to the identification of 261 proteins from CTRL subject UE and 186 from RCC patient UE, and demonstrated that most of the identified proteins are membrane associated or cytoplasmic. Moreover, about a half of identified proteins are not shared between RCC and control UE. Starting from these observations, and from the literature, we selected a panel of 10 proteins, whose UE differential content was subjected to immunoblotting validation. Results show for the first time that RCC UE protein content is substantially and reproducibly different from control UE, and that these differences may provide clues for new RCC biomarker discovery. (hide) EV-METRIC 44% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG (valid.) Protein markers EV: CD9/ Flotilin1/ Syntenin/ TSG101/ CD10/ EMMPRIN/ DPEP1/ CP/ MMP9/ Podocalyxin/ CAIX/ DKK4/ AQP1 non-EV: None Proteomics yes EV density (g/ml) 1.100 TEM measurements 41.1+-12.7 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 60 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ Flotilin1/ Syntenin/ TSG101/ CD10/ EMMPRIN/ DPEP1/ CP/ MMP9/ Podocalyxin/ CAIX/ DKK4/ AQP1 ELISA Detected EV-associated proteins CD10/ EMMPRIN/ DPEP1/ CP/ MMP9/ Podocalyxin/ CAIX/ DKK4/ AQP1 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV130048	1/1	Homo sapiens	Cell culture supernatant	dUC	Pallet N	2013	44%
Study summary Full title A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells All authors Pallet N, Sirois I, Bell C, Hanafi LA, Hamelin K, Dieudé M, Rondeau C, Thibault P, Desjardins M, Hebert MJ Journal Proteomics Abstract The stress status of the apoptotic cell can promote phenotypic changes that have important consequen (show more...)The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1?, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase-dependent manner by serum-starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho-associated, coiled-coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles Membrane(-derived) vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 511.7 (pelleting) Protein markers EV: HSP90/ LAMP2/ VDAC non-EV: Cell organelle protein/ Annexin2 Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 20 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 511.7 Characterization: Protein analysis Western Blot Detected EV-associated proteins HSP90/ LAMP2/ VDAC Detected contaminants Cell organelle protein/ Annexin2 ELISA Detected EV-associated proteins LAMP2/ VDAC Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV130046	1/1	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG	Nanbo A	2013	44%
Study summary Full title Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells All authors Nanbo A, Kawanishi E, Yoshida R, Yoshiyama H Journal J Virol Abstract Epstein-Barr virus (EBV), a human gammaherpesvirus, establishes a lifelong latent infection in B lym (show more...)Epstein-Barr virus (EBV), a human gammaherpesvirus, establishes a lifelong latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence suggest that exosomes derived from EBV-infected cells are internalized and transfer viral factors, including EBV-encoded latent membrane protein and microRNAs, to the recipient cells. However, the detailed mechanism by which exosomes are internalized and their physiological impact on the recipient cells are still poorly understood. In this study, we visualized the internalization of fluorescently labeled exosomes derived from EBV-uninfected and EBV-infected B cells of type I and type III latency into EBV-negative epithelial cells. In this way, we demonstrated that exosomes derived from all three cell types were internalized into the target cells in a similar fashion. Internalization of exosomes was significantly suppressed by treatment with an inhibitor of dynamin and also by the knockdown of caveolin-1. Labeled exosomes were colocalized with caveolae and subsequently trafficked through endocytic pathways. Moreover, we observed that exosomes derived from type III latency cells upregulated proliferation and expression of intercellular adhesion molecule 1 (ICAM-1) in the recipient cells more significantly than did those derived from EBV-negative and type I latency cells. We also identified the EBV latent membrane protein 1 (LMP1) gene as responsible for induction of ICAM-1 expression. Taken together, our data indicate that exosomes released from EBV-infected B cells are internalized via caveola-dependent endocytosis, which, in turn, contributes to phenotypic changes in the recipient cells through transferring one or more viral factors. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG Adj. k-factor 253.9 (pelleting) Protein markers EV: CD63 non-EV: LMP1 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Density gradient Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63 Detected contaminants LMP1 Characterization: Particle analysis

	EV130015	1/1	Homo sapiens	Urine	dUC	Musante L	2013	44%
Study summary Full title Recovery of urinary nanovesicles from ultracentrifugation supernatants All authors Musante L, Saraswat M, Ravidà A, Byrne B, Holthofer H Journal Nephrol Dial Transplant Abstract BACKGROUND: Urinary vesicles represent a newly established source of biological material, widely con (show more...)BACKGROUND: Urinary vesicles represent a newly established source of biological material, widely considered to faithfully represent pathological events in the kidneys and the urogenital epithelium. The majority of currently applied isolation protocols involve cumbersome centrifugation steps to enrich vesicles from urine. To date, the efficiency of these approaches has not been investigated with respect to performing quantitative and qualitative analyses of vesicle populations in the pellet and supernatant (SN) fractions. METHODS: After the series of differential centrifugations, the final SN was reduced to one-twentieth of the original volume by ammonium sulphate precipitation, with the precipitate pellet subjected to another round of differential centrifugations. Electron microscopy, dynamic light scattering and western blot analysis were used to characterize the vesicles present in individual fractions of interest. RESULTS: Pellets obtained after the second set of centrifugations at 200 000 g revealed the presence of vesicles which share a common marker profile, but with distinct differences from those seen in the initial 200 000 g pellet used as the reference. This suggests an enrichment of previously uncharacterized urinary vesicles still in solution after the initial centrifugation steps. Analysis of protein yields recovered post-ultracentrifugation revealed an additional 40% of vesicles retained from the SN. Moreover, these structures showed a formidable resistance to harsh treatments (e.g. 95% ammonium sulphate saturation, hypotonic dialysis, 0.3 M sodium hydroxide). CONCLUSIONS: Methods which employ differential centrifugations of native urine are remarkably ineffective and may lose a substantial population of biologically important vesicle species. (hide) EV-METRIC 44% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles Nano(-sized) vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 78.45 (pelleting) Protein markers EV: CD81/ TSG101/ AQP2/ Podocin/ Beta-actin non-EV: None Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ TSG101/ AQP2/ Podocin/ Beta-actin ELISA Detected EV-associated proteins AQP2/ Podocin/ Beta-actin Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

	EV130014	1/1	Homo sapiens Mus musculus	Cell culture supernatant	dUC Sucrose-DG (valid.)	Li J	2013	44%
Study summary Full title Exosomes mediate the cell-to-cell transmission of IFN-alpha-induced antiviral activity All authors Li J, Liu K, Liu Y, Xu Y, Zhang F, Yang H, Liu J, Pan T, Chen J, Wu M, Zhou X, Yuan Z Journal Nat Immunol Abstract The cell-to-cell transmission of viral resistance is a potential mechanism for amplifying the interf (show more...)The cell-to-cell transmission of viral resistance is a potential mechanism for amplifying the interferon-induced antiviral response. In this study, we report that interferon-? (IFN-?) induced the transfer of resistance to hepatitis B virus (HBV) from nonpermissive liver nonparenchymal cells (LNPCs) to permissive hepatocytes via exosomes. Exosomes from IFN-?-treated LNPCs were rich in molecules with antiviral activity. Moreover, exosomes from LNPCs were internalized by hepatocytes, which mediated the intercellular transfer of antiviral molecules. Finally, we found that exosomes also contributed to the antiviral response of IFN-? to mouse hepatitis virus A59 and adenovirus in mice. Thus, we propose an antiviral mechanism of IFN-? activity that involves the induction and intercellular transfer of antiviral molecules via exosomes. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ CD63/ HSP90/ TSG101/ LAMP2 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.1-1.19 Show all info Study aim Function Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 210000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ HSP90/ TSG101/ LAMP2 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins LAMP2 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV130012	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	Katsuda T	2013	44%
Study summary Full title Human adipose tissue-derived mesenchymal stem cells secrete functional neprilysin-bound exosomes All authors Katsuda T, Tsuchiya R, Kosaka N, Yoshioka Y, Takagaki K, Oki K, Takeshita F, Sakai Y, Kuroda M, Ochiya T Journal Sci Rep Abstract Alzheimer's disease (AD) is characterized by the accumulation of ?-amyloid peptide (A?) in the brain (show more...)Alzheimer's disease (AD) is characterized by the accumulation of ?-amyloid peptide (A?) in the brain because of an imbalance between A? production and clearance. Neprilysin (NEP) is the most important A?-degrading enzyme in the brain. Thus, researchers have explored virus-mediated NEP gene delivery. However, such strategies may entail unexpected risks, and thus exploration of a new possibility for NEP delivery is also required. Here, we show that human adipose tissue-derived mesenchymal stem cells (ADSCs) secrete exosomes carrying enzymatically active NEP. The NEP-specific activity level of 1 ?g protein from ADSC-derived exosomes was equivalent to that of ~ 0.3 ng of recombinant human NEP. Of note, ADSC-derived exosomes were transferred into N2a cells, and were suggested to decrease both secreted and intracellular A? levels in the N2a cells. Importantly, these characteristics were more pronounced in ADSCs than bone marrow-derived mesenchymal stem cells, suggesting the therapeutic relevance of ADSC-derived exosomes for AD. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Protein markers EV: CD63/ CD81 non-EV: Cell organelle protein/ Actin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Wash: volume per pellet (ml) 11 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81 Detected contaminants Cell organelle protein/ Actin Characterization: Particle analysis NTA TRPS EM EM-type cryo EM Image type Close-up

	EV130010	1/1	Homo sapiens	Cell culture supernatant	dUC Iodixanol-DG	Ji H	2013	44%
Study summary Full title Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components All authors Ji H, Greening DW, Barnes TW, Lim JW, Tauro BJ, Rai A, Xu R, Adda C, Mathivanan S, Zhao W, Xue Y, Xu T, Zhu HJ, Simpson RJ Journal Proteomics Abstract Exosomes are small extracellular 40-100 nm diameter membrane vesicles of late endosomal origin that (show more...)Exosomes are small extracellular 40-100 nm diameter membrane vesicles of late endosomal origin that can mediate intercellular transfer of RNAs and proteins to assist premetastatic niche formation. Using primary (SW480) and metastatic (SW620) human isogenic colorectal cancer cell lines we compared exosome protein profiles to yield valuable insights into metastatic factors and signaling molecules fundamental to tumor progression. Exosomes purified using OptiPrep™ density gradient fractionation were 40-100 nm in diameter, were of a buoyant density ~1.09 g/mL, and displayed stereotypic exosomal markers TSG101, Alix, and CD63. A major finding was the selective enrichment of metastatic factors (MET, S100A8, S100A9, TNC), signal transduction molecules (EFNB2, JAG1, SRC, TNIK), and lipid raft and lipid raft-associated components (CAV1, FLOT1, FLOT2, PROM1) in exosomes derived from metastatic SW620 cells. Additionally, using cryo-electron microscopy, ultrastructural components in exosomes were identified. A key finding of this study was the detection and colocalization of protein complexes EPCAM-CLDN7 and TNIK-RAP2A in colorectal cancer cell exosomes. The selective enrichment of metastatic factors and signaling pathway components in metastatic colon cancer cell-derived exosomes contributes to our understanding of the cross-talk between tumor and stromal cells in the tumor microenvironment. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG Protein markers EV: Alix/ CD9/ Flotilin1/ HSP70/ TSG101 non-EV: None Proteomics yes EV density (g/ml) 1.09-1.11 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Density gradient Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 3 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD9/ Flotilin1/ HSP70/ TSG101 Characterization: Particle analysis EM EM-type transmission EM/ cryo EM Image type Close-up, Wide-field

	EV130093	1/1	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG (valid.)	Inuzuka T	2013	44%
Study summary Full title ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells All authors Inuzuka T, Inokawa A, Chen C, Kizu K, Narita H, Shibata H, Maki M Journal Biosci Rep Abstract PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the (show more...)PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activities observed in vitro. Using a previously established cell line of HEK-293 (human embryonic kidney-293) cells constitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 (apoptosis-linked gene 2) Ca²?-dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-BP (2-bromopalmitate, a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich region were necessary for efficient secretion, but ABSs (ALG-2-binding sites) were dispensable. Overexpression of GFP (green fluorescent protein)-fused VPS4B(E235Q), a dominant negative mutant of an AAA (ATPase associated with various cellular activities) ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localized to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP-Rab5A(Q79L)). Secreted Scr3 was taken up by HeLa cells, suggesting that Scr3 functions as a cell-to-cell transferable modulator carried by exosomes in a paracrine manner. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Adj. k-factor 41.45 (pelleting) Protein markers EV: Alix/ Rab5b/ ALG2 non-EV: None Proteomics no EV density (g/ml) 1.100 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type TLA100.2 Pelleting: adjusted k-factor 41.45 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.15 Highest density fraction 2 Orientation Bottom-up Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ Rab5b/ ALG2 ELISA Detected EV-associated proteins Rab5b/ ALG2 Characterization: Particle analysis

	EV130068	1/2	Homo sapiens	Cell culture supernatant	0.2 µm filter Commercial dUC	Hu G	2013	44%
Study summary Full title Release of luminal exosomes contributes to TLR4-mediated epithelial antimicrobial defense All authors Hu G, Gong AY, Roth AL, Huang BQ, Ward HD, Zhu G, Larusso NF, Hanson ND, Chen XM Journal PLoS Pathog Abstract Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. The (show more...)Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Commercial + dUC Adj. k-factor 110.5 (pelleting) Protein markers EV: CD63/ MHC1/ MHC2 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 110.5 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ MHC1/ MHC2 ELISA Detected EV-associated proteins MHC1/ MHC2 Characterization: Particle analysis NTA EM EM-type immune EM/ scanning EM Image type Close-up, Wide-field

	EV130067	2/2	Homo sapiens	Cell culture supernatant	dUC	Hoshino D	2013	44%
Study summary Full title Exosome secretion is enhanced by invadopodia and drives invasive behavior All authors Hoshino D, Kirkbride KC, Costello K, Clark ES, Sinha S, Grega-Larson N, Tyska MJ, Weaver AM Journal Cell Rep Abstract Unconventional secretion of exosome vesicles from multivesicular endosomes (MVEs) occurs across a br (show more...)Unconventional secretion of exosome vesicles from multivesicular endosomes (MVEs) occurs across a broad set of systems and is reported to be upregulated in cancer, where it promotes aggressive behavior. However, regulatory control of exosome secretion is poorly understood. Using cancer cells, we identified specialized invasive actin structures called invadopodia as specific and critical docking and secretion sites for CD63- and Rab27a-positive MVEs. Thus, inhibition of invadopodia formation greatly reduced exosome secretion into conditioned media. Functionally, addition of purified exosomes or inhibition of exosome biogenesis or secretion greatly affected multiple invadopodia life cycle steps, including invadopodia formation, stabilization, and exocytosis of proteinases, indicating a key role for exosome cargoes in promoting invasive activity and providing in situ signaling feedback. Exosome secretion also controlled cellular invasion through three-dimensional matrix. These data identify a synergistic interaction between invadopodia biogenesis and exosome secretion and reveal a fundamental role for exosomes in promoting cancer cell invasiveness. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: CD63/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 180 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV130008	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	Ekström K	2013	44%
Study summary Full title Monocyte exosomes stimulate the osteogenic gene expression of mesenchymal stem cells All authors Ekström K, Omar O, Granéli C, Wang X, Vazirisani F, Thomsen P Journal PLoS One Abstract Inflammation and regeneration at the implant-bone interface are intimately coupled via cell-cell com (show more...)Inflammation and regeneration at the implant-bone interface are intimately coupled via cell-cell communication. In contrast to the prevailing view that monocytes/macrophages orchestrate mesenchymal stem cells (MSCs) and progenitor cells via the secretion of soluble factors, we examined whether communication between these different cell types also occurs via exosomes. LPS-stimulated human monocytes released exosomes, positive for CD9, CD63, CD81, Tsg101 and Hsp70, as determined by flow cytometry and Western blot. These exosomes also contained wide size distribution of RNA, including RNA in the size of microRNAs. The exosomes were shown to interact with human mesenchymal stem cells. After 24 h of culture, a considerable portion of the MSCs had internalised PKH67-labelled exosomes. Furthermore, after 72 h, the gene expression of the osteogenic markers runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein-2 (BMP-2) had increased in comparison with control medium, whereas no significant difference in osteocalcin (OC) expression was demonstrated. The present results show that, under given experimental conditions, monocytes communicate with MSCs via exosomes, resulting in the uptake of exosomes in MSCs and the stimulation of osteogenic differentiation. The present observations suggest that exosomes constitute an additional mode of cell-cell signalling with an effect on MSC differentiation during the transition from injury and inflammation to bone regeneration. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Adj. k-factor 130.7 (pelleting) Protein markers EV: CD63/ CD81/ CD9/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 130.7 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ CD9/ HSP70/ TSG101 Detected contaminants Cell organelle protein Flow cytometry specific beads Selected surface protein(s) Yes Characterization: Particle analysis Particle analysis: flow cytometry

	EV130004	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC UF	Demory Beckler M	2013	44%
Study summary Full title Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS All authors Demory Beckler M, Higginbotham JN, Franklin JL, Ham AJ, Halvey PJ, Imasuen IE, Whitwell C, Li M, Liebler DC, Coffey RJ Journal Mol Cell Proteomics Abstract Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cance (show more...)Activating mutations in KRAS occur in 30% to 40% of colorectal cancers. How mutant KRAS alters cancer cell behavior has been studied intensively, but non-cell autonomous effects of mutant KRAS are less understood. We recently reported that exosomes isolated from mutant KRAS-expressing colon cancer cells enhanced the invasiveness of recipient cells relative to exosomes purified from wild-type KRAS-expressing cells, leading us to hypothesize mutant KRAS might affect neighboring and distant cells by regulating exosome composition and behavior. Herein, we show the results of a comprehensive proteomic analysis of exosomes from parental DLD-1 cells that contain both wild-type and G13D mutant KRAS alleles and isogenically matched derivative cell lines, DKO-1 (mutant KRAS allele only) and DKs-8 (wild-type KRAS allele only). Mutant KRAS status dramatically affects the composition of the exosome proteome. Exosomes from mutant KRAS cells contain many tumor-promoting proteins, including KRAS, EGFR, SRC family kinases, and integrins. DKs-8 cells internalize DKO-1 exosomes, and, notably, DKO-1 exosomes transfer mutant KRAS to DKs-8 cells, leading to enhanced three-dimensional growth of these wild-type KRAS-expressing non-transformed cells. These results have important implications for non-cell autonomous effects of mutant KRAS, such as field effect and tumor progression. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + UF Protein markers EV: Flotilin1/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics yes TEM measurements 59.2+-14.2 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Equal to or above 150,000 g Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV130007	1/2	Homo sapiens	Cell culture supernatant	dUC	Benito-Martin A	2013	44%
Study summary Full title Osteoprotegerin in exosome-like vesicles from human cultured tubular cells and urine All authors Benito-Martin A, Ucero AC, Zubiri I, Posada-Ayala M, Fernandez-Fernandez B, Cannata-Ortiz P, Sanchez-Nino MD, Ruiz-Ortega M, Egido J, Alvarez-Llamas G, Ortiz A Journal PLoS One Abstract Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and rec (show more...)Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles Exosome-like vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 78.45 (pelleting) / 78.45 (washing) Protein markers EV: Alix/ CD63/ TSG101 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Wash: Rotor Type 70Ti Wash: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV130007	2/2	Homo sapiens	Urine	dUC	Benito-Martin A	2013	44%
Study summary Full title Osteoprotegerin in exosome-like vesicles from human cultured tubular cells and urine All authors Benito-Martin A, Ucero AC, Zubiri I, Posada-Ayala M, Fernandez-Fernandez B, Cannata-Ortiz P, Sanchez-Nino MD, Ruiz-Ortega M, Egido J, Alvarez-Llamas G, Ortiz A Journal PLoS One Abstract Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and rec (show more...)Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine. (hide) EV-METRIC 44% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles Exosome-like vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 78.45 (pelleting) Protein markers EV: Alix/ CD63/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV130080	2/2	Homo sapiens	Urine	0.8 µm filter dUC	Barutta F	2013	44%
Study summary Full title Urinary exosomal microRNAs in incipient diabetic nephropathy All authors Barutta F, Tricarico M, Corbelli A, Annaratone L, Pinach S, Grimaldi S, Bruno G, Cimino D, Taverna D, Deregibus MC, Rastaldi MP, Perin PC, Gruden G Journal PLoS One Abstract MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppres (show more...)MicroRNAs (miRNAs), a class of small non-protein-encoding RNAs, regulate gene expression via suppression of target mRNAs. MiRNAs are present in body fluids in a remarkable stable form as packaged in microvesicles of endocytic origin, named exosomes. In the present study, we have assessed miRNA expression in urinary exosomes from type 1 diabetic patients with and without incipient diabetic nephropathy. Results showed that miR-130a and miR-145 were enriched, while miR-155 and miR-424 reduced in urinary exosomes from patients with microalbuminuria. Similarly, in an animal model of early experimental diabetic nephropathy, urinary exosomal miR-145 levels were increased and this was paralleled by miR-145 overexpression within the glomeruli. Exposure of cultured mesangial cells to high glucose increased miR-145 content in both mesangial cells and mesangial cells-derived exosomes, providing a potential mechanism for diabetes-induced miR-145 overexpression. In conclusion, urinary exosomal miRNA content is altered in type 1 diabetic patients with incipient diabetic nephropathy and miR-145 may represent a novel candidate biomarker/player in the complication. (hide) EV-METRIC 44% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.8 µm filter + dUC Adj. k-factor 61.11 (pelleting) / 61.11 (washing) Protein markers EV: Alix/ HSP70/ GAPDH non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 75 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 61.11 Wash: Rotor Type 70.1Ti Wash: adjusted k-factor 61.11 Filtration steps > 0.45 µm, Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ HSP70/ GAPDH Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins GAPDH Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV130028	1/2	Mus musculus	Cell culture supernatant	dUC Iodixanol-DG	An K	2013	44%
Study summary Full title Exosomes neutralize synaptic-plasticity-disrupting activity of Abeta assemblies in vivo All authors An K, Klyubin I, Kim Y, Jung JH, Mably AJ, O'Dowd ST, Lynch T, Kanmert D, Lemere CA, Finan GM, Park JW, Kim TW, Walsh DM, Rowan MJ, Kim JH Journal Mol Brain Abstract BACKGROUND: Exosomes, small extracellular vesicles of endosomal origin, have been suggested to be in (show more...)BACKGROUND: Exosomes, small extracellular vesicles of endosomal origin, have been suggested to be involved in both the metabolism and aggregation of Alzheimer's disease (AD)-associated amyloid ?-protein (A?). Despite their ubiquitous presence and the inclusion of components which can potentially interact with A?, the role of exosomes in regulating synaptic dysfunction induced by A? has not been explored. RESULTS: We here provide in vivo evidence that exosomes derived from N2a cells or human cerebrospinal fluid can abrogate the synaptic-plasticity-disrupting activity of both synthetic and AD brain-derived A?. Mechanistically, this effect involves sequestration of synaptotoxic A? assemblies by exosomal surface proteins such as PrPC rather than A? proteolysis. CONCLUSIONS: These data suggest that exosomes can counteract the inhibitory action of A?, which contributes to perpetual capability for synaptic plasticity. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG Protein markers EV: Alix/ CD81/ Flotilin1/ PrP/ Beta-actin non-EV: None Proteomics no EV density (g/ml) 1.15-1.19 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Density gradient Density medium Iodixanol Lowest density fraction 5 Highest density fraction 30 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD81/ Flotilin1/ PrP/ Beta-actin ELISA Detected EV-associated proteins PrP/ Beta-actin Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV130006	1/1	Mus musculus	Uterine/Oviduct luminal fluid	dUC	Al-Dossary AA	2013	44%
Study summary Full title Expression and secretion of plasma membrane Ca2+-ATPase 4a (PMCA4a) during murine estrus: association with oviductal exosomes and uptake in sperm All authors Al-Dossary AA, Strehler EE, Martin-Deleon PA Journal PLoS One Abstract PMCA4, a membrane protein, is the major Ca(2+) efflux pump in murine sperm where its deletion leads (show more...)PMCA4, a membrane protein, is the major Ca(2+) efflux pump in murine sperm where its deletion leads to a severe loss of hyperactivated motility and to male infertility. We have previously shown that the PMCA4b splice variant interacts with CASK (Ca(2+/)CaM-dependent serine kinase) in regulating sperm Ca(2+). More recently we detected that PMCA4a isoform, in addition to its presence in testis, is secreted in the epididymal luminal fluid and transferred to sperm. Here we show that Pmca4 mRNA is expressed in both the 4a and 4b variants in the vagina, uterus, and oviduct. Immunofluorescence reveals that PMCA4a is similarly expressed and is elevated during estrus, appearing in the glandular and luminal epithelia. Western analysis detected PMCA4a in all tissues and in the luminal fluids (LF) of the vagina (VLF), uterus (ULF), and the oviduct (OLF) collected during estrus. It was ~9- and 4-fold higher in OLF than in VLF and ULF, and only marginally present in LF collected at metestrus/diestrus. Fractionation of the LF collected at estrus, via ultracentrifugation, revealed that 100% of the PMCA4a resides in the vesicular fraction of the ULF and OLF. Transmission electron microscopy (TEM) revealed that OLF vesicles have an exosomal orientation (with the cytoplasmic-side inward), a size range of 25-100 nm, with the characteristic CD9 biomarker. Thus, we dubbed these vesicles oviductosomes, to which PMCA4a was immunolocalized. Incubation of caudal sperm in the combined LF or exosomes resulted in up to a ~3-fold increase of sperm PMCA4a, as detected by flow cytometry, indicating in vitro uptake. Our results are consistent with the increased requirement of Ca(2+) efflux in the oviduct. They show for the first time the presence of oviductal exosomes and highlight their role, along with uterosomes and vaginal exosomes, in post-testicular sperm acquisition of PMCA4a which is essential for hyperactivated motility and fertility. (hide) EV-METRIC 44% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Uterine/Oviduct luminal fluid Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 133 (pelleting) Protein markers EV: CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Uterine/Oviduct luminal fluid Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: rotor type 60Ti Pelleting: adjusted k-factor 133.0 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9 Characterization: Particle analysis EM EM-type transmission EM/ immune EM Image type Close-up, Wide-field

	EV130076	3/3	Gut microbiota	Mouse stool	0.45 µm filter dUC Sucrose-DG (valid.)	Kang CS	2013	43%
Study summary Full title Extracellular vesicles derived from gut microbiota, especially Akkermansia muciniphila, protect the progression of dextran sulfate sodium-induced colitis All authors Kang CS, Ban M, Choi EJ, Moon HG, Jeon JS, Kim DK, Park SK, Jeon SG, Roh TY, Myung SJ, Gho YS, Kim JG, Kim YK Journal PLoS One Abstract Gut microbiota play an important part in the pathogenesis of mucosal inflammation, such as inflammat (show more...)Gut microbiota play an important part in the pathogenesis of mucosal inflammation, such as inflammatory bowel disease (IBD). However, owing to the complexity of the gut microbiota, our understanding of the roles of commensal and pathogenic bacteria in the maintenance of immune homeostasis in the gut is evolving only slowly. Here, we evaluated the role of gut microbiota and their secreting extracellular vesicles (EV) in the development of mucosal inflammation in the gut. Experimental IBD model was established by oral application of dextran sulfate sodium (DSS) to C57BL/6 mice. The composition of gut microbiota and bacteria-derived EV in stools was evaluated by metagenome sequencing using bacterial common primer of 16S rDNA. Metagenomics in the IBD mouse model showed that the change in stool EV composition was more drastic, compared to the change of bacterial composition. Oral DSS application decreased the composition of EV from Akkermansia muciniphila and Bacteroides acidifaciens in stools, whereas increased EV from TM7 phylum, especially from species DQ777900_s and AJ400239_s. In vitro pretreatment of A. muciniphila-derived EV ameliorated the production of a pro-inflammatory cytokine IL-6 from colon epithelial cells induced by Escherichia coli EV. Additionally, oral application of A. muciniphila EV also protected DSS-induced IBD phenotypes, such as body weight loss, colon length, and inflammatory cell infiltration of colon wall. Our data provides insight into the role of gut microbiota-derived EV in regulation of intestinal immunity and homeostasis, and A. muciniphila-derived EV have protective effects in the development of DSS-induced colitis. (hide) EV-METRIC 43% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Mouse stool Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.45 µm filter + dUC + Sucrose-DG (valid.) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function Sample Species Gut microbiota Sample Type Mouse stool Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Pelleting: time(min) 120 Density gradient Only used for validation of main results 1 Density medium Sucrose Speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV130111	1/1	Bos bovis	Milk	0.2 µm filter Density cushion dUC Sucrose-DG	Reinhardt TA	2013	43%
Study summary Full title Bovine milk proteome: quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis All authors Reinhardt TA, Sacco RE, Nonnecke BJ, Lippolis JD Journal J Proteomics Abstract Milk protein expression in healthy cows and cows with mastitis will provide information important fo (show more...)Milk protein expression in healthy cows and cows with mastitis will provide information important for the dairy food industry and immune function in the mammary gland. To facilitate protein discovery, milk was fractioned into whey, milk fat globule membranes (MFGM) and exosomes from healthy and Staphylococcus aureus infected cows. Amine-reactive isobaric tags (iTRAQ) were used to quantify protein changes between milk fractions isolated from healthy and S. aureus infected cows. 2971 milk proteins were identified with a false discovery rate of 0.1%. Greater than 300 milk proteins associated with host defense were identified and 94 were significantly differentially regulated in S. aureus infected milk compared to their uninfected controls. These differentially regulated host defense proteins were selectively segregated in the 3 milk compartments examined. An example of this segregation of host defense proteins was the partitioning and high concentration of proteins indicative of neutrophil extracellular traps (NETs) formation in the MFGM preparations from S. aureus infected milk as compared to exosomes or whey. Protein composition changes found in milk exosomes, MFGM and whey during an infection provides new and comprehensive information on milk protein composition in general as well as changes occurring during an infection.BIOLOGICAL SIGNIFICANCE: The significance of this study is the identification and quantification of the individual components of the neutrophil extracellular traps (NET) functional proteome in an apparent stable complex with MFGM and/or milk fat globules during an intra-mammary infection. NETs could be functionally relevant in intra-mammary infection, as it is known that during an infection neutrophils ingest large amounts of milk fat that down regulates many of their traditional immune functions. Thus the presence of NETs in milk fat provides new insights to mammary immune function and suggests a role for NETs in clinical mastitis. These in vivo NETs can now be tested to determine if they retain functional antimicrobial activity when primarily associated with milk fat. Then we can estimate their real world functional relevance during an intra-mammary infection, which is one key to understanding clinical mastitis in dairy cows. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Density cushion + dUC + Sucrose-DG Adj. k-factor 104.8 (pelleting) Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Bos bovis Sample Type Milk Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 60 Pelleting: rotor type 50.2TI Pelleting: adjusted k-factor 104.8 Density gradient Density medium Sucrose Lowest density fraction 5 Highest density fraction 43 Orientation Bottom-up Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Characterization: Particle analysis

	EV130050	1/1	Shewanella vesiculosa	Bacteria	0.45 µm filter dUC Iodixanol-DG (valid.) UF	Pérez-Cruz C	2013	43%
Study summary Full title New type of outer membrane vesicle produced by the Gram-negative bacterium Shewanella vesiculosa M7T: implications for DNA content All authors Pérez-Cruz C, Carrión O, Delgado L, Martinez G, López-Iglesias C, Mercade E Journal Appl Environ Microbiol Abstract Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA t (show more...)Outer membrane vesicles (OMVs) from Gram-negative bacteria are known to be involved in lateral DNA transfer, but the presence of DNA in these vesicles has remained difficult to explain. An ultrastructural study of the Antarctic psychrotolerant bacterium Shewanella vesiculosa M7(T) has revealed that this Gram-negative bacterium naturally releases conventional one-bilayer OMVs through a process in which the outer membrane is exfoliated and only the periplasm is entrapped, together with a more complex type of OMV, previously undescribed, which on formation drag along inner membrane and cytoplasmic content and can therefore also entrap DNA. These vesicles, with a double-bilayer structure and containing electron-dense material, were visualized by transmission electron microscopy (TEM) after high-pressure freezing and freeze-substitution (HPF-FS), and their DNA content was fluorometrically quantified as 1.8 ± 0.24 ng DNA/?g OMV protein. The new double-bilayer OMVs were estimated by cryo-TEM to represent 0.1% of total vesicles. The presence of DNA inside the vesicles was confirmed by gold DNA immunolabeling with a specific monoclonal IgM against double-stranded DNA. In addition, a proteomic study of purified membrane vesicles confirmed the presence of plasma membrane and cytoplasmic proteins in OMVs from this strain. Our data demonstrate the existence of a previously unobserved type of double-bilayer OMV in the Gram-negative bacterium Shewanella vesiculosa M7(T) that can incorporate DNA, for which we propose the name outer-inner membrane vesicle (O-IMV). (hide) EV-METRIC 43% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bacteria Focus vesicles OMV Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.45 µm filter + dUC + Iodixanol-DG (valid.) + UF Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Omics Sample Species Shewanella vesiculosa Sample Type Bacteria Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting: time(min) 60 Wash: volume per pellet (ml) 25 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 10 Highest density fraction 35 Orientation Bottom-up Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Characterization: Particle analysis EM EM-type transmission EM/ immune EM/ cryo EM Image type Close-up, Wide-field

	EV130045	1/1	Homo sapiens	Urine	dUC	Lv LL	2013	43%
Study summary Full title Isolation and quantification of microRNAs from urinary exosomes/microvesicles for biomarker discovery All authors Lv LL, Cao Y, Liu D, Xu M, Liu H, Tang RN, Ma KL, Liu BC Journal Int J Biol Sci Abstract Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimiz (show more...)Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16 ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×10(5) copies/ul for miR-200c. Exosomes was stable at 4? 24h for shipping before stored at -80? and was stable in urine when stored at -80°C for 12 months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R(2)>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts. (hide) EV-METRIC 43% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles exosomes / microvesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no TEM measurements 30-120 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 60 Characterization: Particle analysis EM EM-type transmission EM/ immune EM Image type Close-up, Wide-field

	EV130065	2/3	Mus musculus	BALF	0.2 µm filter Density cushion (valid.) dUC Immunoaffinity (valid.)	Kulshreshtha A	2013	43%
Study summary Full title Proinflammatory role of epithelial cell-derived exosomes in allergic airway inflammation All authors Kulshreshtha A, Ahmad T, Agrawal A, Ghosh B Journal J Allergy Clin Immunol Abstract BACKGROUND: Exosomes are nanovesicles involved in intercellular communication. Their roles in variou (show more...)BACKGROUND: Exosomes are nanovesicles involved in intercellular communication. Their roles in various diseases are often contextual, depending on the cell type producing them. Although few studies hint toward the proinflammatory role of bronchoalveolar lavage fluid-derived exosomes in asthmatic progression, the cell types in lungs associated with exosome-mediated crosstalk and their resultant effects remain unexplored. OBJECTIVE: It is well established that exosome-mediated cellular communication can influence disease phenotypes. This study explores exosome-mediated cellular crosstalk between structural and immune cells in asthma pathogenesis. METHODS: Exosomes were isolated and detected from bronchoalveolar lavage fluid of control and asthmatic mice and were quantified by using a bead-based assay. Involvement of epithelial cells and macrophages were established by using immunohistochemical techniques in lung tissue sections. The role of IL-13 in exosome production was ascertained by using various in vitro and in vivo techniques. Exosome secretion was blocked in in vitro and in vivo settings by using a chemical inhibitor, and the effects on various asthmatic features were studied. RESULTS: Using combinatorial in vitro and in vivo approaches, we found that exosome secretion and production of exosome-associated proteins are higher in lungs of asthmatic mice compared with that seen in sham mice. Asthma is marked by enhanced secretion of exosomes by epithelial cells, but not macrophages, under the influence of IL-13. These epithelial cell exosomes induce proliferation and chemotaxis of undifferentiated macrophages. On the other hand, GW4869, which inhibited exosome production, resulted in a reduced population of proliferating monocytes and alleviation of various asthmatic features. CONCLUSION: Under the influence of IL-13, epithelial cell-derived exosomes can induce enhanced proliferation and chemotaxis of undifferentiated macrophages in the lungs during asthmatic inflammatory conditions. (hide) EV-METRIC 43% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type BALF Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Density cushion (valid.) + dUC + Immunoaffinity (valid.) Protein markers EV: CD63/ CD81/ HSP70/ MHC2/ Annexin5 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type BALF Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins MHC2/ Annexin5 ELISA Detected EV-associated proteins CD63/ CD81/ HSP70/ MHC2/ Annexin5 Flow cytometry specific beads Selected surface protein(s) Yes Characterization: Particle analysis DLS Particle analysis: flow cytometry EM EM-type transmission EM Image type Close-up, Wide-field

	EV130013	1/1	Homo sapiens	Cell culture supernatant	dUC NS-DG	Kogure T	2013	43%
Study summary Full title Extracellular Vesicle-Mediated Transfer of a Novel Long Noncoding RNA TUC339: A Mechanism of Intercellular Signaling in Human Hepatocellular Cancer All authors Kogure T, Yan IK, Lin WL, Patel T Journal Genes Cancer Abstract Although the expression of long noncoding RNA (lncRNA) is altered in hepatocellular cancer (HCC), th (show more...)Although the expression of long noncoding RNA (lncRNA) is altered in hepatocellular cancer (HCC), their biological effects are poorly defined. We have identified lncRNA with highly conserved sequences, ultraconserved lncRNA (ucRNA) that are transcribed and altered in expression in HCC. Extracellular vesicles, such as exosomes and microvesicles, are released from tumor cells and can transfer biologically active proteins and RNA across cells. We sought to identify the role of vesicle-mediated transfer of ucRNA as a mechanism by which these novel lncRNA could influence intercellular signaling with potential for environmental modulation of tumor cell behavior. HCC-derived extracellular vesicles could be isolated from cells in culture and taken up by adjacent cells. The expression of several ucRNA was dramatically altered within extracellular vesicles compared to that in donor cells. The most highly significantly expressed ucRNA in HCC cell-derived extracellular vesicles was cloned and identified as a 1,198-bp ucRNA, termed TUC339. TUC339 was functionally implicated in modulating tumor cell growth and adhesion. Suppression of TUC339 by siRNA reduced HCC cell proliferation, clonogenic growth, and growth in soft agar. Thus, intercellular transfer of TUC339 represents a unique signaling mechanism by which tumor cells can promote HCC growth and spread. These findings expand the potential roles of ucRNA in HCC, support the existence of selective mechanisms for lncRNA export from cells, and implicate extracellular vesicle-mediated transfer of lncRNA as a mechanism by which tumor cells can modulate their local cellular environment. Intercellular transfer of functionally active RNA molecules by extracellular vesicles provides a mechanism that enables cells to exert genetic influences on other cells within the microenvironment. (hide) EV-METRIC 43% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + NS-DG Protein markers EV: None non-EV: None Proteomics no TEM measurements 105(median) Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Other Name other isolation method NS-DG Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV130069	2/2	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG	Huan J	2013	43%
Study summary Full title RNA trafficking by acute myelogenous leukemia exosomes All authors Huan J, Hornick NI, Shurtleff MJ, Skinner AM, Goloviznina NA, Roberts CT Jr, Kurre P Journal Cancer Res Abstract Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to d (show more...)Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-of-concept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML. (hide) EV-METRIC 43% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG Protein markers EV: None non-EV: None Proteomics no TEM measurements 60-100 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Density gradient Density medium Sucrose Lowest density fraction 8 Highest density fraction 60 Speed (g) 150000 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

	EV130151	4/4	Homo sapiens	Cell culture supernatant	0.2 µm filter SEC UF	Redzic JS	2013	38%
Study summary Full title Extracellular vesicles secreted from cancer cell lines stimulate secretion of MMP-9, IL-6, TGF-beta1 and EMMPRIN All authors Redzic JS, Kendrick AA, Bahmed K, Dahl KD, Pearson CG, Robinson WA, Robinson SE, Graner MW, Eisenmesser EZ Journal PLoS One Abstract Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell (show more...)Extracellular vesicles (EVs) are key contributors to cancer where they play an integral role in cell-cell communication and transfer pro-oncogenic molecules to recipient cells thereby conferring a cancerous phenotype. Here, we purified EVs using straightforward biochemical approaches from multiple cancer cell lines and subsequently characterized these EVs via multiple biochemical and biophysical methods. In addition, we used fluorescence microscopy to directly show internalization of EVs into the recipient cells within a few minutes upon addition of EVs to recipient cells. We confirmed that the transmembrane protein EMMPRIN, postulated to be a marker of EVs, was indeed secreted from all cell lines studied here. We evaluated the response to EV stimulation in several different types of recipient cells lines and measured the ability of these purified EVs to induce secretion of several factors highly upregulated in human cancers. Our data indicate that purified EVs preferentially stimulate secretion of several proteins implicated in driving cancer in monocytic cells but only harbor limited activity in epithelial cells. Specifically, we show that EVs are potent stimulators of MMP-9, IL-6, TGF-?1 and induce the secretion of extracellular EMMPRIN, which all play a role in driving immune evasion, invasion and inflammation in the tumor microenvironment. Thus, by using a comprehensive approach that includes biochemical, biological, and spectroscopic methods, we have begun to elucidate the stimulatory roles. (hide) EV-METRIC 38% (77th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + SEC + UF Protein markers EV: EMMPRIN non-EV: None Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins EMMPRIN ELISA Detected EV-associated proteins EMMPRIN Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV130027	4/4	Mus musculus	Cell culture supernatant	Gel filtration UF	Hajj GN	2013	38%

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