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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100027	2/2	Leishmania donovani Leishmania mexicana Leishmania major	NAY	(d)(U)C DG	Silverman JM	2010	67%
Study summary Full title An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages All authors Silverman JM, Clos J, de'Oliveira CC, Shirvani O, Fang Y, Wang C, Foster LJ, Reiner NE Journal J Cell Sci Abstract Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors i (show more...)Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Adj. k-factor 54.89 (pelleting) Protein markers EV: HSP90/ HSP70/ EF1A non-EV: Proteomics no EV density (g/ml) 1.08-1.17 Show all info Study aim Function Sample Species Leishmania donovani / Leishmania mexicana / Leishmania major Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA100.3 Pelleting: adjusted k-factor 54.89 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Rotor type TLA100.3 Speed (g) 110000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP90/ HSP70/ EF1A ELISA Antibody details provided? No Detected EV-associated proteins EF1A Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein Hsp70 Image type Close-up, Wide-field

	EV100011	2/2	Homo sapiens	NAY	(d)(U)C DG Filtration IAF	Barreto A	2010	67%
Study summary Full title Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T-cell function All authors Barreto A, Rodríguez LS, Rojas OL, Wolf M, Greenberg HB, Franco MA, Angel J Journal Viral Immunol Abstract Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and danger signals rel (show more...)Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and danger signals released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-?1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-?, because they were reversed by treatment of the T cells with the TGF-?-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL), and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration IAF Adj. k-factor 156.9 (pelleting) Protein markers EV: AChE/ CD63/ MFGE8/ HSC70/ HSP70 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.1-1.18;1.24-1.3 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ AChE/ HSC70/ MFGE8 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins AChE/ HSC70/ MFGE8 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100055	1/1	Leishmania donovani	Protozoa	(d)(U)C DG Filtration	Silverman JM	2010	57%
Study summary Full title Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells All authors Silverman JM, Clos J, Horakova E, Wang AY, Wiesgigl M, Kelly I, Lynn MA, McMaster WR, Foster LJ, Levings MK, Reiner NE Journal J Immunol Abstract We investigated the properties of leishmania exosomes with respect to influencing innate and adaptiv (show more...)We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-? in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-?. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-?, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-?-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-? and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function. (hide) EV-METRIC 57% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Protozoa Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 142.6 (pelleting) / 54.89 (washing) Protein markers EV: non-EV: Proteomics yes EV density (g/ml) 1.05-1.16 Show all info Study aim Function Sample Species Leishmania donovani Sample Type Protozoa Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 142.6 Wash: volume per pellet (ml) 1 Wash: Rotor Type TLA100.3 Wash: adjusted k-factor 54.89 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Rotor type 70Ti Speed (g) 110000 Filtration steps 0.22µm or 0.2µm0.2µm > x > 0.1µm Characterization: Protein analysis Characterization: Particle analysis None

	EV100022	1/1	Rattus norvegicus/rattus	Bile	(d)(U)C DG Filtration	Masyuk AI	2010	57%
Study summary Full title Biliary exosomes influence cholangiocyte regulatory mechanisms and proliferation through interaction with primary cilia All authors Masyuk AI, Huang BQ, Ward CJ, Gradilone SA, Banales JM, Masyuk TV, Radtke B, Splinter PL, LaRusso NF Journal Am J Physiol Gastrointest Liver Physiol Abstract Exosomes are small extracellular vesicles that are thought to participate in intercellular communica (show more...)Exosomes are small extracellular vesicles that are thought to participate in intercellular communication. Recent work from our laboratory suggests that, in normal and cystic liver, exosome-like vesicles accumulate in the lumen of intrahepatic bile ducts, presumably interacting with cholangiocyte cilia. However, direct evidence for exosome-ciliary interaction is limited and the physiological relevance of such interaction remains unknown. Thus, in this study, we tested the hypothesis that biliary exosomes are involved in intercellular communication by interacting with cholangiocyte cilia and inducing intracellular signaling and functional responses. Exosomes were isolated from rat bile by differential ultracentrifugation and characterized by scanning, transmission, and immunoelectron microscopy. The exosome-ciliary interaction and its effects on ERK1/2 signaling, expression of the microRNA, miR-15A, and cholangiocyte proliferation were studied on ciliated and deciliated cultured normal rat cholangiocytes. Our results show that bile contains vesicles identified as exosomes by their size, characteristic saucer-shaped morphology, and specific markers, CD63 and Tsg101. When NRCs were exposed to isolated biliary exosomes, the exosomes attached to cilia, inducing a decrease of the phosphorylated-to-total ERK1/2 ratio, an increase of miR-15A expression, and a decrease of cholangiocyte proliferation. All these effects of biliary exosomes were abolished by the pharmacological removal of cholangiocyte cilia. Our findings suggest that bile contains exosomes functioning as signaling nanovesicles and influencing intracellular regulatory mechanisms and cholangiocyte proliferation through interaction with primary cilia. (hide) EV-METRIC 57% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bile Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 142.6 (pelleting) / 142.6 (washing) Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.12-1.21 Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Bile Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 142.6 Wash: volume per pellet (ml) 1 Wash: Rotor Type 70Ti Wash: adjusted k-factor 142.6 Density gradient Lowest density fraction 5 Highest density fraction 30 Orientation Top-down Rotor type 70Ti Speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis EM EM-type transmission EM/ immune EM/ scanning EM EM protein CD63; Tsg101 Image type Close-up, Wide-field Report size (nm) Not reported

	EV100002	1/1	Mus musculus	NAY	(d)(U)C DG	Wang G	2010	56%
Study summary Full title Cellular prion protein released on exosomes from macrophages binds to Hsp70 All authors Wang G, Zhou X, Bai Y, Zhang Z, Zhao D Journal Acta Biochim Biophys Sin Abstract Prion diseases are infectious and fatal neurodegenerative disorders. The cellular prion protein (PrP (show more...)Prion diseases are infectious and fatal neurodegenerative disorders. The cellular prion protein (PrP(C)) converting into misfolded isoform of prion protein (PrP(Sc)) is responsible for prion disease infection. Immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system. Macrophages were considered associated with the transportation and replication of PrP(Sc). So, understanding the PrP(C) trafficking in macrophages is important to explore the transport mechanism for PrP(Sc). Here, we isolated exosomes from the culture medium of Ana-1 macrophage cell line and investigated the PrP(C) trafficked by exosomes and the interaction of PrP(C) with Hsp70 in secreted exosomes by western blotting, immunoelectron microscopy, and co-immunoprecipitation. The results showed that the isolated vesicles from the culture medium of macrophages were characterized by exosomes and bore PrP(C). And PrP(C) bound to Hsp70 both in intracellular environment and secreted exosomes. In contrast, PrP(C) had no interaction with marker proteins of exosomes, Tag101 and Flotillin-1. These results suggested that PrP(C) present in extracellular space might be externalized through secreted exosomes from macrophages, and Hsp70 may play roles in the process of PrP(C) released via secreted exosomes. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: TSG101/ HSP70/ Flotilin1 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.13-1.16 Show all info Study aim Other/PrP trafficking Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Lowest density fraction 0.25 Highest density fraction 2.25 Orientation Bottom-up Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein Tsg101; Flotillin1 Image type Close-up, Wide-field

	EV100006	1/1	Homo sapiens	BALF	(d)(U)C DG Filtration	Qazi KR	2010	56%
Study summary Full title Proinflammatory exosomes in bronchoalveolar lavage fluid of patients with sarcoidosis All authors Qazi KR, Torregrosa Paredes P, Dahlberg B, Grunewald J, Eklund A, Gabrielsson S Journal Thorax Abstract BACKGROUND: Sarcoidosis is a systemic disease of unknown aetiology characterised by granuloma format (show more...)BACKGROUND: Sarcoidosis is a systemic disease of unknown aetiology characterised by granuloma formation and the presence of interferon ? (IFN?)-producing T cells that cause inflammation and tissue damage in multiple organs, especially the lung. Exosomes are nano-sized immunomodulatory vesicles of endosomal origin released from a diverse range of cells and are also found in physiological fluids including bronchoalveolar lavage fluid (BALF) from healthy individuals. OBJECTIVE: To investigate whether exosomes are enriched in the lungs of patients with sarcoidosis compared with healthy individuals and whether they could contribute to pathogenesis. DESIGN: BALF exosomes from patients with sarcoidosis (n=36) and healthy controls (n=14) were compared by electron microscopy, flow cytometry, western blot analysis and mass spectrometry. BALF exosomes were incubated with autologous peripheral blood mononuclear cells (PBMCs) or the human bronchial epithelial cell line 16HBE14o-. Cytokines were measured by ELISPOT and ELISA. RESULTS: BALF from patients with sarcoidosis showed increased levels of exosomes compared with healthy individuals. Exosomes from patients showed significantly higher expression of MHC class I and II, tetraspanins CD9, CD63 and CD81 as well as neuregulin-1, known to be associated with cancer progression. Furthermore, BALF exosomes from patients induced significantly higher IFN? and interleukin (IL)-13 production in autologous PBMCs compared with healthy individuals and could also stimulate IL-8 production from epithelial cells. CONCLUSION: The results indicate for the first time a role for exosomes in human lung disease with possible contributions to the initiation and progression of inflammation in sarcoidosis. This suggests that exosomes may be a new potential target for the clinical treatment of lung diseases. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type BALF Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: CD63/ CD81/ MUC1/ NRG/ HSP70/ MHC2/ CD9/ MHC1 non-EV: Neuregulin/ CD54/ CD80/ CD40/ CD86/ Beta-actin Proteomics yes EV density (g/ml) 1.09-1.19 Show all info Study aim Function Sample Species Homo sapiens Sample Type BALF Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 130 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ HSP70/ MHC1/ MHC2/ MUC1/ NRG Detected contaminants "CD40/ CD54/ CD80/ CD86/ Neuregulin/ Beta-actin" ELISA Antibody details provided? No Detected EV-associated proteins MHC1/ MHC2/ MUC1/ NRG Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100013	2/2	Homo sapiens	NAY	(d)(U)C DG Filtration UF	Mathivanan S	2010	56%
Study summary Full title Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature All authors Mathivanan S, Lim JW, Tauro BJ, Ji H, Moritz RL, Simpson RJ Journal Mol Cell Proteomics Abstract Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell (show more...)Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration UF Protein markers EV: Alix/ HSP70/ TSG101 non-EV: Proteomics yes EV density (g/ml) 1.1-1.12 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 3 Filtration steps 0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP70/ TSG101 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein CD63 Image type Close-up, Wide-field

	EV100003	1/1	Homo sapiens	NAY	(d)(U)C DG Filtration UF	Feng D	2010	50%
Study summary Full title Cellular internalization of exosomes occurs through phagocytosis All authors Feng D, Zhao WL, Ye YY, Bai XC, Liu RQ, Chang LF, Zhou Q, Sui SF Journal Traffic Abstract Exosomes play important roles in many physiological and pathological processes. However, the exosome (show more...)Exosomes play important roles in many physiological and pathological processes. However, the exosome-cell interaction mode and the intracellular trafficking pathway of exosomes in their recipient cells remain unclear. Here, we report that exosomes derived from K562 or MT4 cells are internalized more efficiently by phagocytes than by non-phagocytic cells. Most exosomes were observed attached to the plasma membrane of non-phagocytic cells, while in phagocytic cells these exosomes were found to enter via phagocytosis. Specifically, they moved to phagosomes together with phagocytic polystyrene carboxylate-modified latex beads (biospheres) and were further sorted into phagolysosomes. Moreover, exosome internalization was dependent on the actin cytoskeleton and phosphatidylinositol 3-kinase, and could be inhibited by the knockdown of dynamin2 or overexpression of a dominant-negative form of dynamin2. Further, antibody pretreatment assays demonstrated that tim4 but not tim1 was involved in exosomes uptake. We also found that exosomes did not enter the internalization pathway involving caveolae, macropinocytosis and clathrin-coated vesicles. Our observation that the cellular uptake of exosomes occurs through phagocytosis has important implications for exosome-cell interactions and the exosome intracellular trafficking pathway. (hide) EV-METRIC 50% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration UF Protein markers EV: TSG101/ HSP70 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.11-1.23 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein Tsg101 Image type Close-up, Wide-field

	EV100010	1/1	Bacillus anthracis	Bacteria	(d)(U)C UF	Rivera J	2010	44%
Study summary Full title Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins All authors Rivera J, Cordero RJ, Nakouzi AS, Frases S, Nicola A, Casadevall A Journal Proc Natl Acad Sci U S A Abstract Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is kn (show more...)Extracellular vesicle production is a ubiquitous process in Gram-negative bacteria, but little is known about such process in Gram-positive bacteria. We report the isolation of extracellular vesicles from the supernatants of Bacillus anthracis, a Gram-positive bacillus that is a powerful agent for biological warfare. B. anthracis vesicles formed at the outer layer of the bacterial cell had double-membrane spheres and ranged from 50 to 150 nm in diameter. Immunoelectron microscopy with mAbs to protective antigen, lethal factor, edema toxin, and anthrolysin revealed toxin components and anthrolysin in vesicles, with some vesicles containing more than one toxin component. Toxin-containing vesicles were also visualized inside B. anthracis-infected macrophages. ELISA and immunoblot analysis of vesicle preparations confirmed the presence of B. anthracis toxin components. A mAb to protective antigen protected macrophages against vesicles from an anthrolysin-deficient strain, but not against vesicles from Sterne 34F2 and Sterne ?T strains, consistent with the notion that vesicles delivered both toxin and anthrolysin to host cells. Vesicles were immunogenic in BALB/c mice, which produced a robust IgM response to toxin components. Furthermore, vesicle-immunized mice lived significantly longer than controls after B. anthracis challenge. Our results indicate that toxin secretion in B. anthracis is, at least, partially vesicle-associated, thus allowing concentrated delivery of toxin components to target host cells, a mechanism that may increase toxin potency. Our observations may have important implications for the design of vaccines, for passive antibody strategies, and provide a previously unexplored system for studying secretory pathways in Gram-positive bacteria. (hide) EV-METRIC 44% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bacteria Sample origin NAY Focus vesicles Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: Anthrax toxin polypeptides non-EV: Proteomics yes TEM measurements 50-200 Show all info Study aim Omics Sample Species Bacillus anthracis Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins "Anthrax toxin polypeptides" ELISA Antibody details provided? No Detected EV-associated proteins "Anthrax toxin polypeptides" Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis DLS EM EM-type transmission EM/ immune EM EM protein Anthrax toxin polypeptides Image type Close-up, Wide-field Report size (nm) 50-200

	EV100014	3/3	Homo sapiens	NAY	(d)(U)C DG	Welton JL	2010	44%
Study summary Full title Proteomics analysis of bladder cancer exosomes All authors Welton JL, Khanna S, Giles PJ, Brennan P, Brewis IA, Staffurth J, Mason MD, Clayton A Journal Mol Cell Proteomics Abstract Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids (show more...)Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: CD81/ TSG101/ CD9/ MHC1 non-EV: Proteomics no EV density (g/ml) 1.13-1.16 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed No Density gradient Lowest density fraction 0.2 Highest density fraction 2.5 Orientation Top-down Rotor type TLA110 Speed (g) 150000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ CD9/ TSG101/ MHC1 ELISA Antibody details provided? No Detected EV-associated proteins MHC1 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100009	1/1	Mus musculus	NAY	(d)(U)C DG	Tamboli IY	2010	44%
Study summary Full title Statins promote the degradation of extracellular amyloid {beta}-peptide by microglia via stimulation of exosome-associated insulin-degrading enzyme (IDE) secretion All authors Tamboli IY, Barth E, Christian L, Siepmann M, Kumar S, Singh S, Tolksdorf K, Heneka MT, Lütjohann D, Wunderlich P, Walter J Journal J Biol Chem Abstract Epidemiological studies indicate that intake of statins decrease the risk of developing Alzheimer di (show more...)Epidemiological studies indicate that intake of statins decrease the risk of developing Alzheimer disease. Cellular and in vivo studies suggested that statins might decrease the generation of the amyloid ?-peptide (A?) from the ?-amyloid precursor protein. Here, we show that statins potently stimulate the degradation of extracellular A? by microglia. The statin-dependent clearance of extracellular A? is mainly exerted by insulin-degrading enzyme (IDE) that is secreted in a nonconventional pathway in association with exosomes. Stimulated IDE secretion and A? degradation were also observed in blood of mice upon peripheral treatment with lovastatin. Importantly, increased IDE secretion upon lovastatin treatment was dependent on protein isoprenylation and up-regulation of exosome secretion by fusion of multivesicular bodies with the plasma membrane. These data demonstrate a novel pathway for the nonconventional secretion of IDE via exosomes. The modulation of this pathway could provide a new strategy to enhance the extracellular clearance of A?. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ IDE/ Actin/ Flotilin1 non-EV: Tubulin/ BiP/ Cell organelle protein Proteomics no TEM measurements 81-120 Show all info Study aim Other/How statins promote the clearance of amyloid-beta peptide Sample Species Mus musculus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ Flotilin1/ IDE/ Actin Detected contaminants Cell organelle protein/ "Tubulin/ BiP" ELISA Antibody details provided? No Detected EV-associated proteins IDE/ Actin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field Report size (nm) 81-120

	EV100028	1/1	Mus musculus	NAY	(d)(U)C DG	Tamai K	2010	44%
Study summary Full title Exosome secretion of dendritic cells is regulated by Hrs, an ESCRT-0 protein All authors Tamai K, Tanaka N, Nakano T, Kakazu E, Kondo Y, Inoue J, Shiina M, Fukushima K, Hoshino T, Sano K, Ueno Y, Shimosegawa T, Sugamura K Journal Biochem Biophys Res Commun Abstract Exosomes are nanovesicles derived from multivesicular bodies (MVBs) in antigen-presenting cells. The (show more...)Exosomes are nanovesicles derived from multivesicular bodies (MVBs) in antigen-presenting cells. The components of the ESCRT (endosomal sorting complex required for transport) pathway are critical for the formation of MVBs, however the relationship between the ESCRT pathway and the secretion of exosomes remains unclear. We here demonstrate that Hrs, an ESCRT-0 protein, is required for fascilitating the secretion of exosomes in dendritic cells (DCs). Ultrastructural analyses showed typical saucer-shaped exosomes in the culture supernatant from both the control and Hrs-depleted DCs. However, the amount of exosome secretion was significantly decreased in Hrs-depleted DCs following stimulations with ovalbumin (OVA) as well as calcium ionophore. Antigen-presentation activity was also suppressed in exsosomes purified from Hrs-depleted DCs, while no alteration in OVA degradation was seen in Hrs-depleted DCs. These data indicated that Hrs is involved in the regulation of antigen-presentation activity through the exosome secretion. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: TSG101/ Tubulin/ VPS4B/ HSP70/ MHC2/ MHC1 non-EV: Hrs Proteomics no EV density (g/ml) 1.14-1.15 Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101/ MHC1/ MHC2/ VPS4B/ Tubulin Detected contaminants Hrs ELISA Antibody details provided? No Detected EV-associated proteins MHC1/ MHC2/ VPS4B/ Tubulin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100008	1/1	Homo sapiens	NAY	(d)(U)C DG	Strauss K	2010	44%
Study summary Full title Exosome secretion ameliorates lysosomal storage of cholesterol in Niemann-Pick type C disease All authors Strauss K, Goebel C, Runz H, Möbius W, Weiss S, Feussner I, Simons M, Schneider A Journal J Biol Chem Abstract Niemann-Pick type C1 disease is an autosomal-recessive lysosomal storage disorder. Loss of function (show more...)Niemann-Pick type C1 disease is an autosomal-recessive lysosomal storage disorder. Loss of function of the npc1 gene leads to abnormal accumulation of free cholesterol and sphingolipids within the late endosomal and lysosomal compartments resulting in progressive neurodegeneration and dysmyelination. Here, we show that oligodendroglial cells secrete cholesterol by exosomes when challenged with cholesterol or U18666A, which induces late endosomal cholesterol accumulation. Up-regulation of exosomal cholesterol release was also observed after siRNA-mediated knockdown of NPC1 and in fibroblasts derived from NPC1 patients and could be reversed by expression of wild-type NPC1. We provide evidence that exosomal cholesterol secretion depends on the presence of flotillin. Our findings indicate that exosomal release of cholesterol may serve as a cellular mechanism to partially bypass the traffic block that results in the toxic lysosomal cholesterol accumulation in Niemann-Pick type C1 disease. Furthermore, we suggest that secretion of cholesterol by exosomes contributes to maintain cellular cholesterol homeostasis. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ TSG101/ CD63/ Flotillin2 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.11-1.16 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ TSG101/ Flotillin2 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Flotillin2 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV100026	3/3	Homo sapiens	Urine	(d)(U)C SEC UF	Rood IM	2010	44%
Study summary Full title Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome All authors Rood IM, Deegens JK, Merchant ML, Tamboer WP, Wilkey DW, Wetzels JF, Klein JB Journal Kidney Int Abstract Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many prote (show more...)Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by size-exclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C SEC UF Adj. k-factor 104.8 (pelleting) Protein markers EV: AQP2 non-EV: Albumin Proteomics yes Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 110 Pelleting: rotor type 45Ti Pelleting: adjusted k-factor 104.8 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins AQP2 Detected contaminants Albumin ELISA Antibody details provided? No Detected EV-associated proteins AQP2 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100007	1/1	Mus musculus	NAY	(d)(U)C UF	Ramachandra L	2010	44%
Study summary Full title Mycobacterium tuberculosis synergizes with ATP to induce release of microvesicles and exosomes containing major histocompatibility complex class II molecules capable of antigen presentation All authors Ramachandra L, Qu Y, Wang Y, Lewis CJ, Cobb BA, Takatsu K, Boom WH, Dubyak GR, Harding CV Journal Infect Immun Abstract Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon (show more...)Major histocompatibility complex class II (MHC-II) molecules are released by murine macrophages upon lipopolysaccharide (LPS) stimulation and ATP signaling through the P2X7 receptor. These studies show that infection of macrophages with Mycobacterium tuberculosis or M. bovis strain BCG enhances MHC-II release in synergy with ATP. Shed MHC-II was contained in two distinct organelles, exosomes and plasma membrane-derived microvesicles, which were both able to present exogenous antigenic peptide to T hybridoma cells. Furthermore, microvesicles from mycobacterium-infected macrophages were able to directly present M. tuberculosis antigen (Ag) 85B(241-256)-I-A(b) complexes that were generated by the processing of M. tuberculosis Ag 85B in infected cells to both M. tuberculosis-specific T hybridoma cells and naïve P25 M. tuberculosis T-cell receptor (TCR)-transgenic T cells. In the presence of prefixed macrophages, exosomes from mycobacterium-infected macrophages provided weak stimulation to M. tuberculosis-specific T hybridoma cells but not naïve P25 T cells. Thus, infection with M. tuberculosis primes macrophages for the increased release of exosomes and microvesicles bearing M. tuberculosis peptide-MHC-II complexes that may generate antimicrobial T-cell responses. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes / microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Adj. k-factor 157.1 (pelleting) Protein markers EV: MHC2/ Rab7/ ICAM1/ CD86 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 900 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins MHC2/ CD86/ ICAM1/ Rab7 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ CD86/ ICAM1/ Rab7 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100005	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Ohshima K	2010	44%
Study summary Full title Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line All authors Ohshima K, Inoue K, Fujiwara A, Hatakeyama K, Kanto K, Watanabe Y, Muramatsu K, Fukuda Y, Ogura S, Yamaguchi K, Mochizuki T Journal PLoS One Abstract BACKGROUND: Exosomes play a major role in cell-to-cell communication, targeting cells to transfer ex (show more...)BACKGROUND: Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene down-regulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well. METHODOLOGY/PRINCIPAL FINDINGS: Exosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity. CONCLUSIONS/SIGNIFICANCE: The enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosomes from AZ-P7a cells may reflect their oncogenic characteristics including tumorigenesis and metastasis. Since let-7 miRNAs generally play a tumor-suppressive role as targeting oncogenes such as RAS and HMGA2, our results suggest that AZ-P7a cells release let-7 miRNAs via exosomes into the extracellular environment to maintain their oncogenesis. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 156.9 (pelleting) / 93.46 (washing) Protein markers EV: Alix/ TSG101/ CD29 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: volume per pellet (ml) 6 Wash: Rotor Type 100Ti Wash: adjusted k-factor 93.46 Filtration steps 0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ TSG101/ CD29 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins CD29 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein CD63 Image type Wide-field

	EV100025	1/2	Rattus norvegicus	BSp73AS	(d)(U)C DG	Nazarenko I	2010	44%
Study summary Full title Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation. All authors Nazarenko I, Rana S, Baumann A, McAlear J, Hellwig A, Trendelenburg M, Lochnit G, Preissner KT, Zöller M Journal J Cell Sci Abstract Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumo (show more...)Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumors and tumor-free tissues. However, little information exists on exosome-endothelial cell (EC) interactions or the proangiogenic role of tetraspanins, which are a constitutive component of exosomes. In this study, we used a rat adenocarcinoma model (AS-Tspan8) to explore the effects of exosomal Tspan8 on angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes, including CD106 and CD49d, which were implicated in exosome-EC binding and EC internalization. We found that EC internalized Tspan8-CD49d complex-containing exosomes. Exosome uptake induced vascular endothelial growth factor (VEGF)-independent regulation of several angiogenesis-related genes, including von Willebrand factor, Tspan8, chemokines CXCL5 and MIF, chemokine receptor CCR1, and, together with VEGF, VEGF receptor 2. EC uptake of Tspan8-CD49d complex-containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Unraveling these new pathways of exosome-initiated EC regulation could provide new options for therapeutic interference with tumor-induced angiogenesis. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Tspan8 overexpression Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: TSG101/ CD49d/ Bag6/ CD71/ CD49c/ CD49b/ HSP90/ CD151/ Mac2BP/ LAMP1/ HSP70/ Tspan8/ CD61/ CD9/ CD106 non-EV: None Proteomics yes Show all info Study aim Function, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells BSp73AS EV-harvesting Medium Serum free medium Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.25 M Highest density fraction 2.0 M Sample volume (mL) 0.2 Orientation Bottom-up (sample migrates upwards) Rotor type SW 41 Ti Speed (g) 150000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 2 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD71, HSP70, HSP90, TSG101, CD151, LAMP1, Tspan8, Bag6, CD49b, CD49c, CD49d, CD61, Mac2BP, CD106 Characterization: RNA analysis Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 5 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up Extra information this is publication from 2010. To that time point no NTA, DLS or further technologies were established

	EV100025	2/2	Rattus norvegicus	BSp73AS	(d)(U)C DG	Nazarenko I	2010	44%
Study summary Full title Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation. All authors Nazarenko I, Rana S, Baumann A, McAlear J, Hellwig A, Trendelenburg M, Lochnit G, Preissner KT, Zöller M Journal J Cell Sci Abstract Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumo (show more...)Tumor-derived exosomes containing the tetraspanin Tspan8 can efficiently induce angiogenesis in tumors and tumor-free tissues. However, little information exists on exosome-endothelial cell (EC) interactions or the proangiogenic role of tetraspanins, which are a constitutive component of exosomes. In this study, we used a rat adenocarcinoma model (AS-Tspan8) to explore the effects of exosomal Tspan8 on angiogenesis. Tspan8 contributed to a selective recruitment of proteins and mRNA into exosomes, including CD106 and CD49d, which were implicated in exosome-EC binding and EC internalization. We found that EC internalized Tspan8-CD49d complex-containing exosomes. Exosome uptake induced vascular endothelial growth factor (VEGF)-independent regulation of several angiogenesis-related genes, including von Willebrand factor, Tspan8, chemokines CXCL5 and MIF, chemokine receptor CCR1, and, together with VEGF, VEGF receptor 2. EC uptake of Tspan8-CD49d complex-containing exosomes was accompanied by enhanced EC proliferation, migration, sprouting, and maturation of EC progenitors. Unraveling these new pathways of exosome-initiated EC regulation could provide new options for therapeutic interference with tumor-induced angiogenesis. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: CD49c/ CD61/ CD49b/ CD151/ Mac2BP/ HSP70/ CD9/ CD106 non-EV: None Proteomics yes Show all info Study aim Function, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells BSp73AS EV-harvesting Medium Serum free medium Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: speed (g) 100000 Wash: time (min) 90 Wash: speed (g) 100000 Density gradient Only used for validation of main results Yes Type Continuous Lowest density fraction 0.25 M Highest density fraction 2.0 M Sample volume (mL) 0.2 Orientation Bottom-up (sample migrates upwards) Rotor type SW 41 Ti Speed (g) 150000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 11 Pelleting: duration (min) 2 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 213.2 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins CD151, CD9, CD49b, CD49c, CD61, Mac2BP, CD106, HSP70 Characterization: RNA analysis Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 5 Characterization: Lipid analysis No Characterization: Particle analysis None Extra information this is publication from 2010. To that time point no NTA, DLS or further technologies were established

	EV100023	1/1	Mus musculus	NAY	(d)(U)C DG	Mathews JA	2010	44%
Study summary Full title CD23 Sheddase A disintegrin and metalloproteinase 10 (ADAM10) is also required for CD23 sorting into B cell-derived exosomes All authors Mathews JA, Gibb DR, Chen BH, Scherle P, Conrad DH Journal J Biol Chem Abstract The low affinity receptor for IgE, CD23, is the natural regulator of IgE synthesis, and understandin (show more...)The low affinity receptor for IgE, CD23, is the natural regulator of IgE synthesis, and understanding both the synthesis and the catabolism of CD23 are, thus, important issues. Membrane CD23 is cleaved by a disintegrin and metalloproteinase 10 (ADAM10) and this cleavage influences the ability of CD23 to regulate IgE. In contrast to the belief that cleavage is a cell surface event, endosomal neutralization with NH(4)Cl was found to dramatically reduce CD23 cleavage, suggesting that the majority of CD23 cleavage occurred subsequent to internalization in the endosomal pathway and not at the cell surface. In line with this, full-length CD23 was shown to be sorted in an ADAM10-dependent manner into exosomes. Greatly increased ADAM10-mediated CD23 cleavage was seen at endosomal pH. Additionally, the stalk region of CD23 was found to interact with ADAM10 and ADAM10 binding of CD23 was found to be protease independent. SPR analysis of the interaction indicated about a 10-fold increase in the R(max) at endosomal pH (pH 5.8) compared with pH 7.4, whereas the affinity of the interaction was not significantly changed. The R(max) change, combined with the increased cleavage at endosomal pH, indicates greater accessibility of the CD23 stalk region for ADAM10 at the lower pH. These results indicate a model where CD23 internalization results in ADAM10-dependent incorporation into exosomes, followed by partial cleavage of CD23 by ADAM10 prior to being released from the cell. The increased cleavage at endosomal pH also has implications for other ADAM10 substrates. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Adj. k-factor 124.9 (pelleting) / 124.9 (washing) Protein markers EV: ADAM10 non-EV: Proteomics no EV density (g/ml) 1.38-1.4 Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type AH650 Pelleting: adjusted k-factor 124.9 Wash: volume per pellet (ml) 5 Wash: Rotor Type AH650 Wash: adjusted k-factor 124.9 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Rotor type AH650 Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins ADAM10 ELISA Antibody details provided? No Detected EV-associated proteins ADAM10 Characterization: Particle analysis None

	EV100020	1/1	Homo sapiens	NAY	(d)(U)C DG	Lee HM	2010	44%
Study summary Full title A membranous form of ICAM-1 on exosomes efficiently blocks leukocyte adhesion to activated endothelial cells All authors Lee HM, Choi EJ, Kim JH, Kim TD, Kim YK, Kang C, Gho YS Journal Biochem Biophys Res Commun Abstract While intercellular adhesion molecule-1 (ICAM-1) is a transmembrane protein, two types of extracellu (show more...)While intercellular adhesion molecule-1 (ICAM-1) is a transmembrane protein, two types of extracellular ICAM-1 have been detected in cell culture supernatants as well as in the serum: a soluble form of ICAM-1 (sICAM-1) and a membranous form of ICAM-1 (mICAM-1) associated with exosomes. Previous observations have demonstrated that sICAM-1 cannot exert potent immune modulatory activity due to its low affinity for leukocyte function-associated antigen-1 (LFA-1) or membrane attack complex-1. In this report, we initially observed that human cancer cells shed mICAM-1(+)-exosomes but were devoid of vascular cell adhesion molecule-1 and E-selectin. We demonstrate that mICAM-1 on exosomes retained its topology similar to that of cell surface ICAM-1, and could bind to leukocytes. In addition, we show that exosomal mICAM-1 exhibits potent anti-leukocyte adhesion activity to tumor necrosis factor-alpha-activated endothelial cells compared to that of sICAM-1. Taken together with previous findings, our results indicate that mICAM-1 on exosomes exhibits potent immune modulatory activity. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: CD81/ E-selectin/ ICAM1/ VCAM non-EV: E-selectin/ LFA1/ VCAM1/ Actin Proteomics no EV density (g/ml) 1.12-1.18 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Density gradient Only used for validation of main results Yes Lowest density fraction 0.5 Highest density fraction 2.5 Orientation Bottom-up Rotor type SW55 Speed (g) 150000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ ICAM1/ VCAM/ E-selectin Detected contaminants "VCAM1/ E-selectin/ LFA1/ Actin" ELISA Antibody details provided? No Detected EV-associated proteins ICAM1/ VCAM/ E-selectin Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein ICAM1 Image type Wide-field

	EV100004	1/1	Mus musculus	NAY	(d)(U)C DG Filtration	Guescini M	2010	44%
Study summary Full title C2C12 myoblasts release micro-vesicles containing mtDNA and proteins involved in signal transduction All authors Guescini M, Guidolin D, Vallorani L, Casadei L, Gioacchini AM, Tibollo P, Battistelli M, Falcieri E, Battistin L, Agnati LF, Stocchi V Journal Exp Cell Res Abstract Micro-vesicles can be released by different cell types and operate as 'safe containers' mediating in (show more...)Micro-vesicles can be released by different cell types and operate as 'safe containers' mediating inter-cellular communication. In this work we investigated whether cultured myoblasts could release exosomes. The reported data demonstrate, for the first time, that C2C12 myoblasts release micro-vesicles as shown by the presence of two exosome markers (Tsg101 and Alix proteins). Using real-time PCR analysis it was shown that these micro-vesicles, like other cell types, carry mtDNA. Proteomic characterization of the released micro-vesicle contents showed the presence of many proteins involved in signal transduction. The bioinformatics assessment of the Disorder Index and Aggregation Index of these proteins suggested that C2C12 micro-vesicles mainly deliver the machinery for signal transduction to target cells rather than key proteins involved in hub functions in molecular networks. The presence of IGFBP-5 in the purified micro-vesicles represents an exception, since this binding protein can play a key role in the modulation of the IGF-1 signalling pathway. In conclusion, the present findings demonstrate that skeletal muscle cells release micro-vesicles, which probably have an important role in the communication processes within skeletal muscles and between skeletal muscles and other organs. In particular, the present findings suggest possible new diagnostic approaches to skeletal muscle diseases. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: Alix/ TSG101 non-EV: Proteomics yes Show all info Study aim Omics Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Wash: volume per pellet (ml) 13 Density gradient Only used for validation of main results Yes Lowest density fraction 0.5 Highest density fraction 2.5 Orientation Bottom-up Rotor type 90Ti Speed (g) 150000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ TSG101 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV100001	1/1	Homo sapiens	Urine	(d)(U)C	Fernández-Llama P	2010	44%
Study summary Full title Tamm-Horsfall protein and urinary exosome isolation All authors Fernández-Llama P, Khositseth S, Gonzales PA, Star RA, Pisitkun T, Knepper MA Journal Kidney Int Abstract Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidn (show more...)Urinary exosomes have been proposed as starting material for discovery of protein biomarkers of kidney disease. Current protocols for their isolation use a two-step differential centrifugation process. Due to their low density, exosomes are expected to remain in the low-speed (17,000 x g) supernatant and to sediment only when the sample is spun at high speed (200,000 x g). Analysis using western blot and electron microscopy found that urinary exosomes are also present in the low-speed pellet entrapped by polymeric Tamm-Horsfall protein, thus diminishing the procedure's reproducibility. Here we show that addition of dithiothreitol to the low-speed pellet disrupted the polymeric network, presumably by reduction of disulfide bonds linking the monomers. This modification shifted the exosomal proteins from the low- to the high-speed pellet. Also, by shifting the Tamm-Horsfall protein to the high-speed pellet, the use of dithiothreitol makes it feasible to use Tamm-Horsfall protein to normalize excretion rates of exosomal proteins in spot urines. We tested this by western blot, and found that there was a high degree of correlation between exosomal proteins and Tamm-Horsfall protein in the high-speed pellet. Since the yield of exosomes by differential centrifugation can be increased by chemical reduction, Tamm-Horsfall protein may be a suitable normalizing variable for urinary exosome studies when quantitative urine collections are not practical. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ HSP70/ TSG101/ AQP2/ CD9 non-EV: Tamm-Horsfall glycoprotein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ HSP70/ TSG101/ AQP2 Detected contaminants Tamm-Horsfall glycoprotein ELISA Antibody details provided? No Detected EV-associated proteins AQP2 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV100017	1/2	Homo sapiens	NAY	(d)(U)C DG IAF	Esser J	2010	44%
Study summary Full title Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration All authors Esser J, Gehrmann U, D'Alexandri FL, Hidalgo-Estévez AM, Wheelock CE, Scheynius A, Gabrielsson S, Rådmark O Journal J Allergy Clin Immunol Abstract BACKGROUND: Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the path (show more...)BACKGROUND: Leukotrienes (LTs) are potent proinflammatory lipid mediators with key roles in the pathogenesis of asthma and inflammation. Recently, nanovesicles (exosomes), released from macrophages and dendritic cells (DCs), have become increasingly appreciated as messengers in immunity. OBJECTIVE: We investigated whether exosomes from human macrophages, DCs, and plasma contain enzymes for LT biosynthesis and studied potential roles for exosomes in transcellular LT metabolism and granulocyte chemotaxis. METHODS: The presence of LT pathway enzymes and LT biosynthesis in exosomes and cells was analyzed by Western blot, immunoelectron microscopy, and enzyme activity assays. Surface marker expression was evaluated by flow cytometry, and granulocyte migration was assessed in a multiwell chemotaxis system. RESULTS: Exosomes from macrophages and DCs contain functional enzymes for LT biosynthesis. After incubation of intact cells with the LT biosynthesis intermediate LTA(4), LTB(4) was the major product of macrophages, whereas DCs primarily formed LTC(4). However, in exosomes from both cell types, LTC(4) was the predominant LTA(4) metabolite. Exosomal LTC(4) formation (per milligram protein) exceeded that of cells. In macrophages and DCs, TGF-?1 upregulated LTA(4) hydrolase along with increased LTB(4) formation also in the exosomes. Moreover, TGF-?1 modified the expression of surface marker proteins on cells and exosomes and reduced the exosome yield from macrophages. On Ca(2+)-ionophore and arachidonic acid stimulation, exosomes produced chemotactic eicosanoids and induced granulocyte migration. Interestingly, active LTA(4) hydrolase and LTC(4) synthase were present also in exosomes from human plasma. CONCLUSION: Our findings indicate that exosomes can contribute to inflammation by participation in LT biosynthesis and granulocyte recruitment. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG IAF Protein markers EV: CD81/ CD63/ Beta-actin/ MHC2 non-EV: Proteomics no EV density (g/ml) 1.13-1.19 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 100000 Immunoaffinity capture Selected surface protein(s) MHC2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ MHC2/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ Beta-actin Characterization: Particle analysis EM EM-type immune EM EM protein LTC4S Image type Close-up

	EV100035	1/1	Homo sapiens	NAY	(d)(U)C DG IAF	Buschow SI	2010	44%
Study summary Full title MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis All authors Buschow SI, van Balkom BW, Aalberts M, Heck AJ, Wauben M, Stoorvogel W Journal Immunol Cell Biol Abstract Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) car (show more...)Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of this compartment with the plasma membrane. We have previously shown that in contrast to the sorting of MHC II at lysosomally targeted multivesicular bodies, sorting of MHC II into exosomes does not rely on MHC II ubiquitination. In search for proteins that drive the incorporation of MHC II into exosomes or functionally discriminate exosomal from plasma membrane MHC II, we first analyzed the total proteome of highly purified B cell-derived exosomes using sensitive and accurate mass spectrometry (MS), and identified 539 proteins, including known and not previously identified constituents. Using quantitative MS, we then identified a small subset of proteins that were specifically co-immunoprecipitated with MHC II from detergent-solubilized exosomes. These include HSC71, HSP90, 14-3-3?, CD20 and pyruvate kinase type M2 (PKM2), and we speculate on the functionality of their interaction with exosomal MHC II. (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG IAF Adj. k-factor 362.8 (pelleting) Protein markers EV: CD81/ MHC2 non-EV: Proteomics yes EV density (g/ml) 1.150 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 362.8 Density gradient Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Immunoaffinity capture Selected surface protein(s) MHC2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ MHC2 ELISA Antibody details provided? No Detected EV-associated proteins MHC2 Characterization: Particle analysis None

	EV100014	1/3	Homo sapiens	NAY	(d)(U)C DC	Welton JL	2010	38%
Study summary Full title Proteomics analysis of bladder cancer exosomes All authors Welton JL, Khanna S, Giles PJ, Brennan P, Brewis IA, Staffurth J, Mason MD, Clayton A Journal Mol Cell Proteomics Abstract Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids (show more...)Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Protein markers EV: TSG101/ CD63/ CD81/ HSP90/ GAPDH/ LAMP2/ LAMP1/ CD9/ MHC1 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ HSP90/ TSG101/ MHC1/ LAMP1/ LAMP2/ GAPDH Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MHC1/ LAMP1/ LAMP2/ GAPDH Characterization: Particle analysis None

	EV100019	1/1	Homo sapiens	NAY	DG IAF	Hurwitz MD	2010	38%
Study summary Full title Radiation therapy induces circulating serum Hsp72 in patients with prostate cancer All authors Hurwitz MD, Kaur P, Nagaraja GM, Bausero MA, Manola J, Asea A Journal Radiother Oncol Abstract BACKGROUND AND PURPOSE: Hsp72 found in the extracellular milieu has been shown to play an important (show more...)BACKGROUND AND PURPOSE: Hsp72 found in the extracellular milieu has been shown to play an important role in immune regulation. The impact of common cancer therapies on extracellular release of Hsp72 however, has been to date undefined. MATERIALS AND METHODS: Serum from 13 patients undergoing radiation therapy (XRT) for prostate cancer with or without hormonal therapy (ADT) was measured for levels of circulating serum Hsp72 and pro-inflammatory cytokines (IL-6 and TNF-alpha) using the classical sandwich ELISA technique and the relative expression of CD8(+) T lymphocytes and natural killer (NK) cells was measured using flow cytometry. Mouse orthotopic xenograft of human prostate cancer tumors (DU-145 and PC-3) were used to validate and further characterize the response noted in the clinical setting. The biological significance of tumor released Hsp72 was studied in human dendritic cells (DC) in vitro. RESULTS: Circulating serum Hsp72 levels increased an average of 3.5-fold (median per patient 4.8-fold) with XRT but not with ADT (p=0.0002). Increases in IL-6 (3.3-fold), TNF-alpha (1.8-fold), CD8(+) CTL (2.1-fold) and NK cells (3.2-fold) also occurred. Using PC-3 and DU-145 human prostate cancer xenograft models in mice, we confirmed that XRT induces Hsp72 release primarily from implanted tumors. In vitro studies using supernatant recovered from irradiated human prostate cancer cells point to exosomes containing Hsp72 as a possible stimulator of pro-inflammatory cytokine production and costimulatory molecules expression in human DC. CONCLUSIONS: The current study confirms for the first time in an actual clinical setting elevation of circulating serum Hsp72 with XRT. The accompanying studies in mice and in vitro identify the released exosomes containing Hsp72 as playing a pivotal role in stimulating pro-inflammatory immune responses. These findings, if validated, may lead to new treatment paradigms for common human malignancies. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG IAF Protein markers EV: Tubulin/ HSC73 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method Density gradient Lowest density fraction 1.08g/L Highest density fraction 1.25g/L Orientation Top-down Immunoaffinity capture Selected surface protein(s) anti-human MHC class II Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Tubulin/ HSC73 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Tubulin/ HSC73 Characterization: Particle analysis None

	EV100031	1/1	Homo sapiens	NAY	(d)(U)C Filtration IAF	Zumaquero E	2010	33%
Study summary Full title Exosomes from human lymphoblastoid B cells express enzymatically active CD38 that is associated with signaling complexes containing CD81, Hsc-70 and Lyn All authors Zumaquero E, Muñoz P, Cobo M, Lucena G, Pavón EJ, Martín A, Navarro P, García-Pérez A, Ariza-Veguillas A, Malavasi F, Sancho J, Zubiaur M Journal Exp Cell Res Abstract Exosome vesicles of endocytic origin are involved in communication between tumor and immune cells. I (show more...)Exosome vesicles of endocytic origin are involved in communication between tumor and immune cells. In addition, membrane rafts (MR) may support the sorting of proteins associated with exosomes. CD38 is found at the plasma membrane and in recycling endosomes, which are both redistributed toward the immunological synapse (IS) upon T cell antigen receptor (TCR) engagement. The data of this study provide evidence that CD38 is expressed on the surface of secreted exosomes derived from lymphoblastoid B cells. Exosomic CD38 is associated with the signaling molecules CD81, Hsc-70 and Lyn. Likewise, in MR, CD38 is associated with CD81, CD19, Lyn, Galphai-2, Hsc-70 and actin. Therefore, a high degree of overlap in the pattern of signaling proteins associated with CD38 in exosomes and MR exists. Exosomic and MR CD38, by virtue of these interactions, have signaling potential. Indeed, CD38 is enzymatically active in both exosomes and MR, and CD38 ligation induces Akt/PKB and Erk activation, which is accompanied by increased translocation of CD38 into MR. In conclusion, the present study indicates that CD38 localizes to MR, where it promotes cell signaling, and it is exported out of the cells through the exosome-mediated exocytic pathway, where it may act as an intercellular messenger. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration IAF Adj. k-factor 124.9 (pelleting) / 124.9 (washing) Protein markers EV: CD38/ Rab5/ Hsc70/ CD81/ Lyn/ MHC2 non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 180-720 Pelleting: rotor type AH650 Pelleting: adjusted k-factor 124.9 Wash: Rotor Type AH650 Wash: adjusted k-factor 124.9 Filtration steps 0.22µm or 0.2µm Immunoaffinity capture Selected surface protein(s) CD38, Lyn, CD81, Hsc70 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ MHC2/ CD38/ Rab5/ Lyn/ Hsc70 ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ CD38/ Rab5/ Lyn/ Hsc70 Characterization: Particle analysis EM EM-type immune EM EM protein CD38 Image type Close-up

	EV100029	1/1	Homo sapiens	NAY	(d)(U)C DG Filtration	Temme S	2010	33%
Study summary Full title The herpes simplex virus-1 encoded glycoprotein B diverts HLA-DR into the exosome pathway All authors Temme S, Eis-Hübinger AM, McLellan AD, Koch N Journal J Immunol Abstract Neutralizing Abs play an important role for immunity against HSV-1 infection. This branch of the imm (show more...)Neutralizing Abs play an important role for immunity against HSV-1 infection. This branch of the immune response is initiated by MHC class II Ag presentation and activation of T cell help. In this study, we show that the HSV-1 encoded glycoprotein B (gB) manipulates the class II processing pathway by perturbing endosomal sorting and trafficking of HLA-DR (DR) molecules. Expression of gB in the human melanoma cell line Mel JuSo results in formation of enlarged DR(+) intracellular vesicles. Costaining of the vesicles revealed the presence of DR, gB, and the late endosomal marker CD63. The lumen of these late endosomal membranes shows a variable content, containing either gB or CD63, or both CD63 and gB. gB targets DR molecules on their biosynthetic route, after the MHC class II invariant chain is released from the DR heterodimer. gB-DR complexes were detected in a post-Golgi compartment and in exosomes, but not on the cell surface. Interestingly, increasing expression of gB strongly elevated the amount of DR and CD63 released into the exosome pathway. In conclusion, this is a previously undescribed mode of viral immune evasion involving hijacking of DR from its normal transport route to the cell surface, followed by viral-mediated release of DR into the exosome pathway. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: CD63 non-EV: Cell organelle protein Proteomics no 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Wash: volume per pellet (ml) 1 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Detected contaminants Cell organelle protein Characterization: Particle analysis None

	EV100027	1/2	Leishmania donovani Leishmania mexicana Leishmania major	NAY	(d)(U)C DC UF	Silverman JM	2010	33%
Study summary Full title An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages All authors Silverman JM, Clos J, de'Oliveira CC, Shirvani O, Fang Y, Wang C, Foster LJ, Reiner NE Journal J Cell Sci Abstract Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors i (show more...)Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC UF Adj. k-factor 54.89 (pelleting) Protein markers EV: HSP90/ HSP70/ EF1A non-EV: Proteomics yes Show all info Study aim Function Sample Species Leishmania donovani / Leishmania mexicana / Leishmania major Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA100.3 Pelleting: adjusted k-factor 54.89 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP90/ HSP70/ EF1A ELISA Antibody details provided? No Detected EV-associated proteins EF1A Characterization: Particle analysis None

	EV100026	1/3	Homo sapiens	Urine	(d)(U)C	Rood IM	2010	33%
Study summary Full title Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome All authors Rood IM, Deegens JK, Merchant ML, Tamboer WP, Wilkey DW, Wetzels JF, Klein JB Journal Kidney Int Abstract Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many prote (show more...)Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by size-exclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 104.8 (pelleting) Protein markers EV: AQP2 non-EV: Albumin Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 110 Pelleting: rotor type 45Ti Pelleting: adjusted k-factor 104.8 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins AQP2 Detected contaminants Albumin ELISA Antibody details provided? No Detected EV-associated proteins AQP2 Characterization: Particle analysis None

	EV100047	1/1	Homo sapiens	NAY	(d)(U)C	Merendino AM	2010	33%
Study summary Full title Hsp60 is actively secreted by human tumor cells All authors Merendino AM, Bucchieri F, Campanella C, Marcianò V, Ribbene A, David S, Zummo G, Burgio G, Corona DF, Conway de Macario E, Macario AJ, Cappello F Journal PLoS One Abstract BACKGROUND: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular ch (show more...)BACKGROUND: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. METHODOLOGY/PRINCIPAL FINDINGS: Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. CONCLUSIONS/SIGNIFICANCE: Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 145.1 (pelleting) Protein markers EV: Alix/ AChE non-EV: Proteomics no Show all info Study aim Other/HSP60 secretion Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 60Ti Pelleting: adjusted k-factor 145.1 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ AChE ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100046	2/3	Homo sapiens	Urine	(d)(U)C	Merchant ML	2010	33%
Study summary Full title Microfiltration isolation of human urinary exosomes for characterization by MS All authors Merchant ML, Powell DW, Wilkey DW, Cummins TD, Deegens JK, Rood IM, McAfee KJ, Fleischer C, Klein E, Klein JB Journal Proteomics Clin Appl Abstract PURPOSE: The purpose of this study was to address the hypothesis that small vesicular urinary partic (show more...)PURPOSE: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein-binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies. EXPERIMENTAL DESIGN: We characterized a microfiltration approach using a low protein binding, hydrophilized polyvinylidene difluoride membrane to easily and efficiently isolate urinary exosomes from fresh, room temperature or 4°C urine, with a simultaneous depletion of abundant urinary proteins. Using LC-MS, immunoblot analysis, and electron microscopy methods, we demonstrate this method to isolate intact exosomes and thereby enrich for a low abundant urinary proteome. RESULTS: In comparison to other standard methods of exosome isolation including ultracentrifugation and nanofiltration, we demonstrate equivalent enrichment of the exosome proteome with reduced co-purification of abundant urinary proteins. CONCLUSION AND CLINICAL RELEVANCE: In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 104.8 (pelleting) Protein markers EV: AQP2/ NHE3/ CD10 non-EV: Albumin Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 110 Pelleting: rotor type 45Ti Pelleting: adjusted k-factor 104.8 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD10/ NHE3/ AQP2 Detected contaminants Albumin ELISA Antibody details provided? No Detected EV-associated proteins CD10/ NHE3/ AQP2 Characterization: Particle analysis None

	EV100021	1/2	Homo sapiens	NAY	(d)(U)C Filtration	Lenassi M	2010	33%
Study summary Full title HIV Nef is secreted in exosomes and triggers apoptosis in bystander CD4+ T cells All authors Lenassi M, Cagney G, Liao M, Vaupotic T, Bartholomeeusen K, Cheng Y, Krogan NJ, Plemenitas A, Peterlin BM Journal Traffic Abstract The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed (show more...)The HIV accessory protein negative factor (Nef) is one of the earliest and most abundantly expressed viral proteins. It is also found in the serum of infected individuals (Caby MP, Lankar D, Vincendeau-Scherrer C, Raposo G, Bonnerot C. Exosomal-like vesicles are present in human blood plasma. Int Immunol 2005;17:879-887). Extracellular Nef protein has deleterious effects on CD4(+) T cells (James CO, Huang MB, Khan M, Garcia-Barrio M, Powell MD, Bond VC. Extracellular Nef protein targets CD4(+) T cells for apoptosis by interacting with CXCR4 surface receptors. J Virol 2004;78:3099-3109), the primary targets of HIV, and can suppress immunoglobulin class switching in bystander B cells (Qiao X, He B, Chiu A, Knowles DM, Chadburn A, Cerutti A. Human immunodeficiency virus 1 Nef suppresses CD40-dependent immunoglobulin class switching in bystander B cells. Nat Immunol 2006;7:302-310). Nevertheless, the mode of exit of Nef from infected cells remains a conundrum. We found that Nef stimulates its own export via the release of exosomes from all cells examined. Depending on its intracellular location, these Nef exosomes form at the plasma membrane, late endosomes or both compartments in Jurkat, SupT1 and primary T cells, respectively. Nef release through exosomes is conserved also during HIV-1 infection of peripheral blood lymphocytes (PBLs). Released Nef exosomes cause activation-induced cell death of resting PBLs in vitro. Thus, HIV-infected cells export Nef in bioactive vesicles, which facilitate the depletion of CD4(+) T cells that is a hallmark of acquired immunodeficiency syndrome (AIDS). (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ HSC70/ CD81/ Alix/ Annexin2/ LAMP2/ ICAM1/ AChE/ Beta-actin/ CD9/ MHC1 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ CD81/ CD9/ TSG101/ HSC70/ LAMP2/ AChE/ Beta-actin/ Annexin2/ ICAM1/ MHC1 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins HSC70/ LAMP2/ AChE/ Beta-actin/ Annexin2/ ICAM1/ MHC1 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV100042	1/1	Canis familiaris	NAY	(d)(U)C DG	Komatsu T	2010	33%
Study summary Full title Extrusion of Na,K-ATPase and transferrin receptor with lipid raft-associated proteins in different populations of exosomes during reticulocyte maturation in dogs All authors Komatsu T, Arashiki N, Otsuka Y, Sato K, Inaba M Journal Jpn J Vet Res Abstract The present study characterizes canine reticulocyte exosomes. Exosomes are small membrane vesicles i (show more...)The present study characterizes canine reticulocyte exosomes. Exosomes are small membrane vesicles involved in membrane remodeling that are released from reticulocytes during the final maturation step of red blood cells. The vesicles collected from reticulocyte culture supernatants by differential centrifugation contained major exosomal proteins including heat shock protein cognate 70 (Hsc70) and transferrin receptors (TfR), consistent with the definition of the exosome. In addition, the Na,K-ATPase alpha-subunit and stomatin, a lipid raft-associated protein, were extruded by the exosome pathway, possibly leading to the absence of these proteins in erythrocytes, while the major protein constituents of erythrocyte membranes, spectrin and band 3 were retained in reticulocytes and not expelled into exosomes. The Na,K-ATPase alpha-subunit, as well as TfR and about half of the stomatin contained in exosomes, was obtained in a detergent-soluble fraction that was distinct from the lipid raft microdomain. Moreover, Na,K-ATPase and a portion of stomatin were distributed differently to Hsc70, TfR, stomatin, and ganglioside GM1 in vesicles separated with sucrose density gradient centrifugation. These results demonstrate that a heterogeneous group of exosomes participates in the loss of Na,K-ATPase and membrane remodeling during reticulocyte maturation in dogs. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: HSC70/ Flotillin2 non-EV: Proteomics yes EV density (g/ml) 1.06-1.14 Show all info Study aim Biogenesis/Sorting Sample Species Canis familiaris Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 10 Highest density fraction 35 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSC70/ Flotillin2 ELISA Antibody details provided? No Detected EV-associated proteins HSC70/ Flotillin2 Characterization: Particle analysis None

	EV100066	2/2	Bos bovis	Milk	(d)(U)C DG	Hata T	2010	33%
Study summary Full title Isolation of bovine milk-derived microvesicles carrying mRNAs and microRNAs All authors Hata T, Murakami K, Nakatani H, Yamamoto Y, Matsuda T, Aoki N Journal Biochem Biophys Res Commun Abstract By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approxima (show more...)By a series of centrifugation and ultracentrifugation, we could isolate microvesicles with approximately 100 nm in diameter from bovine milk. We also found that approximately 1700 and 1000 ng of total RNA, in which small RNAs were major components, was contained inside the microvesicles isolated from 6 ml of colostrum and mature milk, respectively, despite high RNase activity in the milk. Polyadenylated gene transcripts for major milk proteins and translation elongation factor-1alpha (EF-1alpha) were present in the microvesicles, and integrity of some transcripts was confirmed by real-time PCR targeting 5'- and 3'-ends of mRNA and by in vitro translation analysis. Moreover, a considerable amount of mammary gland and immune-related microRNAs were present in the milk-derived microvesicles. Acidification of milk to mimic gastrointestinal tract did not mostly affected RNA yield and quality. The milk related gene transcripts were detected in cultured cells when incubated with milk-derived microvesicles, suggesting cellular uptake of the microvesicle contents including RNA. Our findings suggest that bovine breast milk contains RNAs capable for being transferred to living cells and involved in the development of calf's gastrointestinal and immune systems. (hide) EV-METRIC 33% (53rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Adj. k-factor 255.8 (pelleting) Protein markers EV: MFGE8 non-EV: Proteomics no EV density (g/ml) 1.16-1.2 Show all info Study aim Function Sample Species Bos bovis Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins MFGE8 ELISA Antibody details provided? No Detected EV-associated proteins MFGE8 Characterization: Particle analysis EM EM-type transmission EM/ scanning EM Image type Close-up Report size (nm) Not reported

	EV100018	1/1	Homo sapiens Mus musculus	NAY	(d)(U)C DC	Gourzones C	2010	33%
Study summary Full title Extra-cellular release and blood diffusion of BART viral micro-RNAs produced by EBV-infected nasopharyngeal carcinoma cells All authors Gourzones C, Gelin A, Bombik I, Klibi J, Vérillaud B, Guigay J, Lang P, Témam S, Schneider V, Amiel C, Baconnais S, Jimenez AS, Busson P Journal Virol J Abstract BACKGROUND: Nasopharyngeal carcinoma (NPC) is a human epithelial malignancy consistently associated (show more...)BACKGROUND: Nasopharyngeal carcinoma (NPC) is a human epithelial malignancy consistently associated with the Epstein-Barr virus. The viral genome is contained in the nuclei of all malignant cells with abundant transcription of a family of viral microRNAs called BART miRNAs. MicroRNAs are well known intra-cellular regulatory elements of gene expression. In addition, they are often exported in the extra-cellular space and sometimes transferred in recipient cells distinct from the producer cells. Extra-cellular transport of the microRNAs is facilitated by various processes including association with protective proteins and packaging in secreted nanovesicles called exosomes. Presence of microRNAS produced by malignant cells has been reported in the blood and saliva of tumor-bearing patients, especially patients diagnosed with glioblastoma or ovarian carcinoma. In this context, it was decided to investigate extra-cellular release of BART miRNAs by NPC cells and their possible detection in the blood of NPC patients. To address this question, we investigated by quantitative RT-PCR the status of 5 microRNAs from the BART family in exosomes released by NPC cells in vitro as well as in plasma samples from NPC xenografted nude mice and NPC patients. RESULTS: We report that the BART miRNAs are released in the extra-cellular space by NPC cells being associated, at least to a large extent, with secreted exosomes. They are detected with a good selectivity in plasma samples from NPC xenografted nude mice as well as NPC patients. CONCLUSIONS: Viral BART miRNAs are secreted by NPC cells in vitro and in vivo. They have enough stability to diffuse from the tumor site to the peripheral blood. This study provides a basis to explore their potential as a source of novel tumor biomarkers and their possible role in communications between malignant and non-malignant cells. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Adj. k-factor 317.8 (pelleting) Protein markers EV: Beta-actin/ CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 45Ti Pelleting: adjusted k-factor 317.8 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Beta-actin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100012	1/2	Mus musculus	Urine	(d)(U)C DC Filtration	Conde-Vancells J	2010	33%
Study summary Full title Candidate biomarkers in exosome-like vesicles purified from rat and mouse urine samples All authors Conde-Vancells J, Rodriguez-Suarez E, Gonzalez E, Berisa A, Gil D, Embade N, Valle M, Luka Z, Elortza F, Wagner C, Lu SC, Mato JM, Falcon-Perez M Journal Proteomics Clin Appl Abstract PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of he (show more...)PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of hepatic disease in order to inform the diagnosis, prognosis and treatment. EXPERIMENTAL DESIGN: We have investigated the proteome of urinary vesicles present in urine samples obtained from experimental models for the study of liver injury, as an approach for identifying potential biomarkers for hepatic disease. RESULTS: The biochemical and proteomic characterization of highly purified exosome-like urinary vesicles has identified 28 proteins previously unreported in these vesicles, and many that have been previously associated with diseases, such as the prion-related protein. Furthermore, in urine samples from D-galactosamine-treated rats, a well-characterized experimental model for acute liver injury, we have detected a severe reduction in some proteins that normally are clearly detected in urinary vesicles. Finally, differential protein content on urinary vesicles from a mouse model for chronic liver injury has been also identified. CONCLUSIONS AND CLINICAL RELEVANCE: Our results argue positively that urinary vesicles could be a source for identifying non-invasive biomarkers of liver injury. We proposed some proteins such as Cd26, Cd81, Slc3A1 and Cd10 that have been found to be differentially expressed in urinary vesicles from some of the analyzed models as potential biomarkers for liver injury. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles Exosome-like vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration Protein markers EV: TSG101/ CD63/ LimpII/ AQP1/ CD81/ Slc3a1/ Flotillin/ CD26/ PrP/ CD10/ HSP70 non-EV: Tamm-Horsfall glycoprotein Proteomics yes TEM measurements 95.8+-50.7 Show all info Study aim Biomarker Sample Species Mus musculus Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ HSP70/ TSG101/ CD26/ CD10/ LimpII/ PrP/ Slc3a1/ AQP1/ Flotillin Detected contaminants Tamm-Horsfall glycoprotein ELISA Antibody details provided? No Detected EV-associated proteins CD26/ CD10/ LimpII/ PrP/ Slc3a1/ AQP1/ Flotillin Characterization: Particle analysis EM EM-type cryo EM Image type Close-up Report size (nm) 95.8+-50.7

	EV100012	2/2	Rattus norvegicus/rattus	Urine	(d)(U)C DG Filtration	Conde-Vancells J	2010	33%
Study summary Full title Candidate biomarkers in exosome-like vesicles purified from rat and mouse urine samples All authors Conde-Vancells J, Rodriguez-Suarez E, Gonzalez E, Berisa A, Gil D, Embade N, Valle M, Luka Z, Elortza F, Wagner C, Lu SC, Mato JM, Falcon-Perez M Journal Proteomics Clin Appl Abstract PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of he (show more...)PURPOSE: There is a compelling clinical imperative to identify discerning molecular biomarkers of hepatic disease in order to inform the diagnosis, prognosis and treatment. EXPERIMENTAL DESIGN: We have investigated the proteome of urinary vesicles present in urine samples obtained from experimental models for the study of liver injury, as an approach for identifying potential biomarkers for hepatic disease. RESULTS: The biochemical and proteomic characterization of highly purified exosome-like urinary vesicles has identified 28 proteins previously unreported in these vesicles, and many that have been previously associated with diseases, such as the prion-related protein. Furthermore, in urine samples from D-galactosamine-treated rats, a well-characterized experimental model for acute liver injury, we have detected a severe reduction in some proteins that normally are clearly detected in urinary vesicles. Finally, differential protein content on urinary vesicles from a mouse model for chronic liver injury has been also identified. CONCLUSIONS AND CLINICAL RELEVANCE: Our results argue positively that urinary vesicles could be a source for identifying non-invasive biomarkers of liver injury. We proposed some proteins such as Cd26, Cd81, Slc3A1 and Cd10 that have been found to be differentially expressed in urinary vesicles from some of the analyzed models as potential biomarkers for liver injury. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles Exosome-like vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: TSG101/ Caveolin/ CD63/ CD81/ Slc3a1/ Flotillin/ CD10 non-EV: Proteomics no EV density (g/ml) 1.18;1.246;1.252 Show all info Study aim Biomarker Sample Species Rattus norvegicus/rattus Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Rotor type TLA110 Speed (g) 210000 Pelleting-wash: volume per pellet (mL) 2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101/ CD10/ Slc3a1/ Caveolin/ Flotillin ELISA Antibody details provided? No Detected EV-associated proteins CD10/ Slc3a1/ Caveolin/ Flotillin Characterization: Particle analysis EM EM-type cryo EM Image type Wide-field

	EV100016	1/1	Mus musculus	NAY	(d)(U)C	Bulloj A	2010	33%
Study summary Full title Insulin-degrading enzyme sorting in exosomes: a secretory pathway for a key brain amyloid-beta degrading protease All authors Bulloj A, Leal MC, Xu H, Castaño EM, Morelli L Journal J Alzheimers Dis Abstract The accumulation of amyloid-beta (Abeta) peptides in senile plaques is one of the hallmarks of Alzhe (show more...)The accumulation of amyloid-beta (Abeta) peptides in senile plaques is one of the hallmarks of Alzheimer's disease (AD) progression. The endocytic pathway has been proposed as a major subcellular site for Abeta generation while the compartments in which Abeta-degrading proteases interact with Abeta are still elusive. It was suggested that extracellular Abeta degradation may take place by plasma-membrane associated proteases or by extracellular proteases, among which insulin-degrading enzyme (IDE) is the most relevant. However, the mechanisms of IDE secretion are poorly understood. In the present study we used N2a cells to explore if IDE is indeed released through exosomes and the effect of exosomes release on extracellular levels of Abeta. We demonstrated that proteolytically-active plasma membrane associated-IDE is routed in living N2a cells to multivesicular bodies and subsequently, a major fraction is sorted to exosomes. We described that extracellular IDE levels decrease if the generation of multivesicular bodies is interfered and may be positively modulated by exosomes release under stress-induced conditions. Our results reinforce the relevance of functional IDE in the catabolism of extracellular Abeta. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: HSP70/ IDE/ Flotilin1 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ HSP70/ IDE Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins IDE Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein IDE; Tf-receptor Image type Close-up, Wide-field

	EV100034	1/1	Rattus norvegicus/rattus	NAY	(d)(U)C DG	Barrès C	2010	33%
Study summary Full title Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages All authors Barrès C, Blanc L, Bette-Bobillo P, André S, Mamoun R, Gabius HJ, Vidal M Journal Blood Abstract Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocy (show more...)Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Tf-receptor/ Actin/ LAMP2 non-EV: Proteomics no EV density (g/ml) 1.08-1.18 Show all info Study aim Biogenesis/Sorting Sample Species Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 0.5 Highest density fraction 2.5 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Tf-receptor/ LAMP2/ Actin ELISA Antibody details provided? No Detected EV-associated proteins Tf-receptor/ LAMP2/ Actin Characterization: Particle analysis None

	EV100062	1/1	Saccharomyces cerevisiae	Yeast	(d)(U)C Filtration UF	Oliveira DL	2010	29%
Study summary Full title Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis All authors Oliveira DL, Nakayasu ES, Joffe LS, Guimarães AJ, Sobreira TJ, Nosanchuk JD, Cordero RJ, Frases S, Casadevall A, Almeida IC, Nimrichter L, Rodrigues ML Journal PLoS One Abstract BACKGROUND: Extracellular vesicles in yeast cells are involved in the molecular traffic across the c (show more...)BACKGROUND: Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells. CONCLUSIONS/SIGNIFICANCE: Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells. (hide) EV-METRIC 29% (58th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Yeast Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: non-EV: Proteomics yes Show all info Study aim Biogenesis/Sorting Sample Species Saccharomyces cerevisiae Sample Type Yeast Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps > 0.45 µm, Characterization: Protein analysis Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV100058	1/1	Mus musculus	NAY	(d)(U)C DG	Xiang X	2010	29%
Study summary Full title TLR2-mediated expansion of MDSCs is dependent on the source of tumor exosomes All authors Xiang X, Liu Y, Zhuang X, Zhang S, Michalek S, Taylor DD, Grizzle W, Zhang HG Journal Am J Pathol Abstract Exosomes released from tumor cells having been shown to induce interleukin-6 release from myeloid-de (show more...)Exosomes released from tumor cells having been shown to induce interleukin-6 release from myeloid-derived suppressor cells in a Toll-like receptor 2/Stat3-dependent manner. In this study, we show that exosomes released from tumor cells re-isolated from syngeneic mice are capable of inducing interleukin-6 in a Toll-like receptor 2-independent manner, whereas the data generated from exosomes of tumor cells having undergone numerous in vitro passages induce interleukin-6 in a Toll-like receptor 2-dependent manner. This discrepancy may be due to the source of tumor cells used to generate the exosomes for this study. These results suggest that exosomes released from tumor cells that are not within a tumor microenvironment may not realistically represent the role of tumor exosomes in vivo. This is an important consideration since frequently passing tumor cells in vivo is an accepted practice for studying tumor exosome-mediated inflammatory responses. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Adj. k-factor 362.8 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 362.8 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 70000 Characterization: Particle analysis None

	EV100070	5/5	Homo sapiens	Urine	(d)(U)C DG Filtration	Miranda KC	2010	29%
Study summary Full title Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease All authors Miranda KC, Bond DT, McKee M, Skog J, Panescu TG, Da Silva N, Brown D, Russo LM Journal Kidney Int Abstract Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers (show more...)Urinary exosomes or microvesicles are being studied intensively to identify potential new biomarkers for renal disease. We sought to identify whether these microvesicles contain nucleic acids. We isolated microvesicles from human urine in the same density range as that previously described for urinary exosomes and found them to have an RNA integrity profile similar to that of kidney tissue, including 18S and 28S rRNA. This profile was better preserved in urinary microvesicles compared with whole cells isolated from urine, suggesting that microvesicles may protect RNA during urine passage. We were able to detect mRNA in the human urinary microvesicles encoding proteins from all regions of the nephron and the collecting duct. Further, to provide a proof of principle, we found that microvesicles isolated from the urine of the V-ATPase B1 subunit knockout mice lacked mRNA of this subunit while containing a normal amount of the B2 subunit and aquaporin 2. The microvesicles were found to be contaminated with extraneous DNA potentially on their surface; therefore, we developed a rapid and reliable means to isolate nucleic acids from within urine microvesicles devoid of this extraneous contamination. Our study provides an experimental strategy for the routine isolation and use of urinary microvesicles as a novel and non-invasive source of nucleic acids to further renal disease biomarker discovery. (hide) EV-METRIC 29% (60th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes / microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.06-1.09 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Density gradient Only used for validation of main results Yes Orientation Top-down Speed (g) 110000 Pelleting-wash: volume per pellet (mL) 20 Filtration steps > 0.45 µm, Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV100044	1/1	Mus musculus	NAY	(d)(U)C DG	Liu Y	2010	29%
Study summary Full title Contribution of MyD88 to the tumor exosome-mediated induction of myeloid derived suppressor cells All authors Liu Y, Xiang X, Zhuang X, Zhang S, Liu C, Cheng Z, Michalek S, Grizzle W, Zhang HG Journal Am J Pathol Abstract In this study we observed that mice pretreated with tumor exosomes had a significant acceleration of (show more...)In this study we observed that mice pretreated with tumor exosomes had a significant acceleration of tumor metastasis in the lung. Tumor metastasis correlated significantly with an increase in recruitment of more Myeloid-derived suppressor cells (MDSCs) in the lung of C57BL/6j (B6) mice pretreated with tumor exosomes. These effects were blunted when MyD88 knockout (KO) mice were pretreated with tumor exosomes. MDSCs induced by tumor exosomes and isolated from wild-type B6 mice also more potently inhibited T cell activation and induction of interleukin-6 and tumor necrosis factor-alpha than MDSCs isolated from the lung of MyD88 KO mice. In vitro, addition of tumor exosomes to bone marrow-derived CD11b(+)Gr-1(+) cells isolated from wild-type B6 mice resulted in more cytokine production, including tumor necrosis factor-alpha, interleukin-6, and the chemokine CCL2, than CD11b(+)Gr-1(+) cells isolated from MyD88 KO mice. Moreover, lower levels of CCL2 were observed in the lungs in MyD88 KO mice pretreated with tumor exosomes than that in wild-type mice. Together these data demonstrate a pivotal role for MyD88 in tumor exosome-mediated expansion of MDSCs and tumor metastasis. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Adj. k-factor 362.8 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 362.8 Density gradient Lowest density fraction 0.25 Highest density fraction 2.5 Speed (g) 70000 Characterization: Particle analysis None

	EV100080	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Klein-Scory S	2010	29%
Study summary Full title Immunoscreening of the extracellular proteome of colorectal cancer cells All authors Klein-Scory S, Kübler S, Diehl H, Eilert-Micus C, Reinacher-Schick A, Stühler K, Warscheid B, Meyer HE, Schmiegel W, Schwarte-Waldhoff I Journal BMC Cancer Abstract BACKGROUND: The release of proteins from tumors can trigger an immune response in cancer patients in (show more...)BACKGROUND: The release of proteins from tumors can trigger an immune response in cancer patients involving T lymphocytes and B lymphocytes, which results in the generation of antibodies to tumor-derived proteins. Many studies aim to use humoral immune responses, namely autoantibody profiles, directly, as clinical biomarkers. Alternatively, the antibody immune response as an amplification system for tumor associated alterations may be used to indicate putative protein biomarkers with high sensitivity. Aiming at the latter approach we here have implemented an autoantibody profiling strategy which particularly focuses on proteins released by tumor cells in vitro: the so-called secretome. METHODS: For immunoscreening, the extracellular proteome of five colorectal cancer cell lines was resolved on 2D gels, immobilized on PVDF membranes and used for serological screening with individual sera from 21 colorectal cancer patients and 24 healthy controls. All of the signals from each blot were assigned to a master map, and autoantigen candidates were defined based of the pattern of immunoreactivities. The corresponding proteins were isolated from preparative gels, identified by MALDI-MS and/or by nano-HPLC/ESI-MS/MS and exemplarily confirmed by duplex Western blotting combining the human serum samples with antibodies directed against the protein(s) of interest. RESULTS: From 281 secretome proteins stained with autoantibodies in total we first defined the background patterns of frequently immunoreactive extracellular proteins in healthy and diseased people. An assignment of these proteins, among them many nominally intracellular proteins, to the subset of exosomal proteins within the secretomes revealed a large overlap. On this basis we defined and consequently confirmed novel biomarker candidates such as the extreme C-terminus of the extracellular matrix protein agrin within the set of cancer-enriched immunoreactivities. CONCLUSIONS: Our findings suggest, first, that autoantibody responses may be due, in large part, to cross-presentation of antigens to the immune system via exosomes, membrane vesicles released by tumor cells and constituting a significant fraction of the secretome. In addition, this immunosecretomics approach has revealed novel biomarker candidates, some of them secretome-specific, and thus serves as a promising complementary tool to the frequently reported immunoproteomic studies for biomarker discovery. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 105.3 (pelleting) Protein markers EV: non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type T890 Pelleting: adjusted k-factor 105.3 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Characterization: Particle analysis None

	EV100026	2/3	Homo sapiens	Urine	(d)(U)C UF	Rood IM	2010	25%
Study summary Full title Comparison of three methods for isolation of urinary microvesicles to identify biomarkers of nephrotic syndrome All authors Rood IM, Deegens JK, Merchant ML, Tamboer WP, Wilkey DW, Wetzels JF, Klein JB Journal Kidney Int Abstract Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many prote (show more...)Urinary microvesicles, such as 40-100 nm exosomes and 100-1000 nm microparticles, contain many proteins that may serve as biomarkers of renal disease. Microvesicles have been isolated by ultracentrifugation or nanomembrane ultrafiltration from normal urine; however, little is known about the efficiency of these methods in isolating microvesicles from patients with nephrotic-range proteinuria. Here we compared three techniques to isolate microvesicles from nephrotic urine: nanomembrane ultrafiltration, ultracentrifugation, and ultracentrifugation followed by size-exclusion chromatography (UC-SEC). Highly abundant urinary proteins were still present in sufficient quantity after ultrafiltration or ultracentrifugation to blunt detection of less abundant microvesicular proteins by MALDI-TOF-TOF mass spectrometry. The microvesicular markers neprilysin, aquaporin-2, and podocalyxin were highly enriched following UC-SEC compared with preparations by ultrafiltration or ultracentrifugation alone. Electron microscopy of the UC-SEC fractions found microvesicles of varying size, compatible with the presence of both exosomes and microparticles. Thus, UC-SEC following ultracentrifugation to further enrich and purify microparticles facilitates the search for prognostic biomarkers that might be used to predict the clinical course of nephrotic syndrome. (hide) EV-METRIC 25% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: AQP2 non-EV: Albumin Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed No Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins AQP2 Detected contaminants Albumin ELISA Antibody details provided? No Detected EV-associated proteins AQP2 Characterization: Particle analysis None

	EV100046	1/3	Homo sapiens	Urine	(d)(U)C UF	Merchant ML	2010	25%
Study summary Full title Microfiltration isolation of human urinary exosomes for characterization by MS All authors Merchant ML, Powell DW, Wilkey DW, Cummins TD, Deegens JK, Rood IM, McAfee KJ, Fleischer C, Klein E, Klein JB Journal Proteomics Clin Appl Abstract PURPOSE: The purpose of this study was to address the hypothesis that small vesicular urinary partic (show more...)PURPOSE: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein-binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies. EXPERIMENTAL DESIGN: We characterized a microfiltration approach using a low protein binding, hydrophilized polyvinylidene difluoride membrane to easily and efficiently isolate urinary exosomes from fresh, room temperature or 4°C urine, with a simultaneous depletion of abundant urinary proteins. Using LC-MS, immunoblot analysis, and electron microscopy methods, we demonstrate this method to isolate intact exosomes and thereby enrich for a low abundant urinary proteome. RESULTS: In comparison to other standard methods of exosome isolation including ultracentrifugation and nanofiltration, we demonstrate equivalent enrichment of the exosome proteome with reduced co-purification of abundant urinary proteins. CONCLUSION AND CLINICAL RELEVANCE: In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput. (hide) EV-METRIC 25% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: AQP2/ NHE3/ CD10 non-EV: Albumin Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD10/ NHE3/ AQP2 Detected contaminants Albumin ELISA Antibody details provided? No Detected EV-associated proteins CD10/ NHE3/ AQP2 Characterization: Particle analysis None

	EV100046	3/3	Homo sapiens	Urine	(d)(U)C Filtration UF	Merchant ML	2010	25%
Study summary Full title Microfiltration isolation of human urinary exosomes for characterization by MS All authors Merchant ML, Powell DW, Wilkey DW, Cummins TD, Deegens JK, Rood IM, McAfee KJ, Fleischer C, Klein E, Klein JB Journal Proteomics Clin Appl Abstract PURPOSE: The purpose of this study was to address the hypothesis that small vesicular urinary partic (show more...)PURPOSE: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein-binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies. EXPERIMENTAL DESIGN: We characterized a microfiltration approach using a low protein binding, hydrophilized polyvinylidene difluoride membrane to easily and efficiently isolate urinary exosomes from fresh, room temperature or 4°C urine, with a simultaneous depletion of abundant urinary proteins. Using LC-MS, immunoblot analysis, and electron microscopy methods, we demonstrate this method to isolate intact exosomes and thereby enrich for a low abundant urinary proteome. RESULTS: In comparison to other standard methods of exosome isolation including ultracentrifugation and nanofiltration, we demonstrate equivalent enrichment of the exosome proteome with reduced co-purification of abundant urinary proteins. CONCLUSION AND CLINICAL RELEVANCE: In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput. (hide) EV-METRIC 25% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: Ezrin/ AQP2/ Neprilysin/ LAMP2/ CD10/ NHE3 non-EV: Albumin Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD10/ NHE3/ AQP2/ LAMP2/ Neprilysin/ Ezrin Detected contaminants Albumin ELISA Antibody details provided? No Detected EV-associated proteins CD10/ NHE3/ AQP2/ LAMP2/ Neprilysin/ Ezrin Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein Neprilysin;M6P receptor;LAMP2; Tf-receptor Image type Wide-field

	EV100045	1/1	Homo sapiens	NAY	(d)(U)C DC Filtration UF	Medina A	2010	25%
Study summary Full title Transdifferentiated circulating monocytes release exosomes containing 14-3-3 proteins with matrix metalloproteinase-1 stimulating effect for dermal fibroblasts All authors Medina A, Ghahary A Journal Wound Repair Regen Abstract Fibroblasts are major cellular components of healing wounds. In this regard, it remains to be fully (show more...)Fibroblasts are major cellular components of healing wounds. In this regard, it remains to be fully understood how different paracrine signals may influence the final collagen/matrix metalloproteinase (MMP) balance in resident fibroblasts. Our previous reports have demonstrated that circulating stem cells and monocytes can be transdifferentiated into keratinocyte-like cells under certain culture conditions. These transformed cells are able to stimulate MMP-1 expression in dermal fibroblasts. However, the underlying mechanism of this cell-to-cell interaction is unknown. This study describes exosomes as a major delivery system that keratinocyte-like cells use to release proteins into the conditioned media. The exosomes exhibited distinctive size, density, and saucer-like morphology. Using PKH-26 and GFP-adenovirus infection, we demonstrated that exosomes are able to fuse and then release their protein content into dermal fibroblasts. Mass spectrometry and Western blotting identified five 14-3-3 isoforms (beta, gamma, epsilon, tau, and zeta) as MMP-1 stimulating factors for dermal fibroblasts. Immunoprecipation assays confirmed that these 14-3-3 isoforms account for almost the entire MMP-1 up-regulation induced by exosomes. In summary, our results demonstrated that circulating monocytes stimulated to be transformed into keratinocyte-like cells could promote an anti-fibrogenic commitment of dermal fibroblasts via exosomal 14-3-3 proteins. (hide) EV-METRIC 25% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration UF Protein markers EV: Beta-actin/ HSC70 non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps > 0.45 µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSC70/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins HSC70/ Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV100013	1/2	Homo sapiens	NAY	(d)(U)C Filtration IAF UF	Mathivanan S	2010	25%
Study summary Full title Proteomics analysis of A33 immunoaffinity-purified exosomes released from the human colon tumor cell line LIM1215 reveals a tissue-specific protein signature All authors Mathivanan S, Lim JW, Tauro BJ, Ji H, Moritz RL, Simpson RJ Journal Mol Cell Proteomics Abstract Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell (show more...)Exosomes are 40-100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers. (hide) EV-METRIC 25% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration IAF UF Protein markers EV: TSG101/ HSP70 non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.2µm > x > 0.1µm Immunoaffinity capture Selected surface protein(s) A33 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP70/ TSG101 Characterization: Particle analysis None

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