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Results list Most used isolation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV140047	2/2	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	Gonzalez E	2014	44%
Study summary Full title Human mammospheres secrete hormone-regulated active extracellular vesicles All authors Gonzalez E, Piva M, Rodriguez-Suarez E, Gil D, Royo F, Elortza F, Falcon-Perez JM, Vivanco Md Journal PLoS One Abstract Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important pro (show more...)Breast cancer is a leading cause of cancer-associated death worldwide. One of the most important prognostic factors for survival is the early detection of the disease. Recent studies indicate that extracellular vesicles may provide diagnostic information for cancer management. We demonstrate the secretion of extracellular vesicles by primary breast epithelial cells enriched for stem/progenitor cells cultured as mammospheres, in non-adherent conditions. Using a proteomic approach we identified proteins contained in these vesicles whose expression is affected by hormonal changes in the cellular environment. In addition, we showed that these vesicles are capable of promoting changes in expression levels of genes involved in epithelial-mesenchymal transition and stem cell markers. Our findings suggest that secreted extracellular vesicles could represent potential diagnostic and/or prognostic markers for breast cancer and support a role for extracellular vesicles in cancer progression. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Protein markers EV: CD63/ CD81/ Flotilin1/ MFGE8/ CD13/ CD133/ Annexin2 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 serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ Flotilin1/ MFGE8/ CD13/ CD133/ Annexin2 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins MFGE8/ CD13/ CD133/ Annexin2 Fluorescent NTA Characterization: Particle analysis NTA EM EM-type cryo EM Image type Close-up

	EV140111	1/2	Homo sapiens	Cell culture supernatant	dUC	Cypryk W	2014	44%
Study summary Full title Quantitative proteomics of extracellular vesicles released from human monocyte-derived macrophages upon beta-glucan stimulation All authors Cypryk W, Ohman T, Eskelinen EL, Matikainen S, Nyman TA Journal J Proteome Res Abstract Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially (show more...)Fungal infections (mycoses) are common diseases of varying severity that cause problems, especially to immunologically compromised people. Fungi express a variety of pathogen-associated molecular patterns on their surface including ?-glucans, which are important immunostimulatory components of fungal cell walls. During stimulatory conditions of infection and colonization, besides intensive intracellular response, human cells actively communicate on the intercellular level by secreting proteins and other biomolecules with several mechanisms. Vesicular secretion remains one of the most important paths for the proteins to exit the cell. Here, we have used high-throughput quantitative proteomics combined with bioinformatics to characterize and quantify vesicle-mediated protein release from ?-glucan-stimulated human macrophages differentiated in vitro from primary blood monocytes. We show that ?-glucan stimulation induces vesicle-mediated protein secretion. Proteomic study identified 540 distinct proteins from the vesicles, and the identified proteins show a proteomic signature characteristic for their cellular origin. Importantly, we identified several receptors, including cation-dependent mannose-6-phosphate receptor, macrophage scavenger receptor, and P2X7 receptor, that have not been identified from vesicles before. Proteomic data together with detailed pathway and network analysis showed that integrins and their cytoplasmic cargo proteins are highly abundant in extracellular vesicles released upon ?-glucan stimulation. In conclusion, the present data provides a solid basis for further studies on the functional role of vesicular protein secretion upon fungal infection. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 295 (pelleting) Protein markers EV: Alix/ TSG101/ Annexin1/ Tubulin non-EV: None Proteomics no Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 295.0 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ TSG101/ Annexin1/ Tubulin ELISA Detected EV-associated proteins Annexin1/ Tubulin Fluorescent NTA Characterization: Particle analysis NTA EM EM-type transmission EM/ immune EM Image type Close-up, Wide-field

	EV140110	2/2	Mus musculus	Cell culture supernatant	dUC Sucrose-DG	Cossetti C	2014	44%
Study summary Full title Extracellular vesicles from neural stem cells transfer IFN-gamma via Ifngr1 to activate Stat1 signaling in target cells All authors Cossetti C, Iraci N, Mercer TR, Leonardi T, Alpi E, Drago D, Alfaro-Cervello C, Saini HK, Davis MP, Schaeffer J, Vega B, Stefanini M, Zhao C, Muller W, Garcia-Verdugo JM, Mathivanan S, Bachi A, Enright AJ, Mattick JS, Pluchino S Journal Mol Cell Abstract The idea that stem cell therapies work only via cell replacement is challenged by the observation of (show more...)The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-?) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-? bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-?/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-?/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG Adj. k-factor 122.2 (pelleting) / 89.21 (washing) Protein markers EV: Alix/ CD63/ CD9/ HSP90/ HSP70/ TSG101/ Ago1/ Ago2/ Tnfr1/ Beta-actin non-EV: None Proteomics no EV density (g/ml) 1.13-1.20 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 122.2 Wash: Rotor Type TLA55 Wash: adjusted k-factor 89.21 Density gradient Density medium Sucrose Lowest density fraction 0.32 Highest density fraction 2 Orientation Top-down Rotor type TLA55 Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ CD9/ HSP90/ HSP70/ TSG101/ Ago1/ Ago2/ Tnfr1/ Beta-actin ELISA Detected EV-associated proteins Ago1/ Ago2/ Tnfr1/ Beta-actin Fluorescent NTA Characterization: Particle analysis NTA

	EV140108	1/1	Acholeplasma laidlawii	Mycoplasma	0.1 µm filter dUC Iodixanol-DG UF	Chernov VM	2014	44%
Study summary Full title Extracellular membrane vesicles secreted by mycoplasma Acholeplasma laidlawii PG8 are enriched in virulence proteins All authors Chernov VM, Mouzykantov AA, Baranova NB, Medvedeva ES, Grygorieva TY, Trushin MV, Vishnyakov IE, Sabantsev AV, Borchsenius SN, Chernova OA Journal J Proteomics Abstract Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Arc (show more...)Mycoplasmas (class Mollicutes), the smallest prokaryotes capable of self-replication, as well as Archaea, Gram-positive and Gram-negative bacteria constitutively produce extracellular vesicles (EVs). However, little is known regarding the content and functions of mycoplasma vesicles. Here, we present for the first time a proteomics-based characterisation of extracellular membrane vesicles from Acholeplasma laidlawii PG8. The ubiquitous mycoplasma is widespread in nature, found in humans, animals and plants, and is the causative agent of phytomycoplasmoses and the predominant contaminant of cell cultures. Taking a proteomics approach using LC-ESI-MS/MS, we identified 97 proteins. Analysis of the identified proteins indicated that A. laidlawii-derived EVs are enriched in virulence proteins that may play critical roles in mycoplasma-induced pathogenesis. Our data will help to elucidate the functions of mycoplasma-derived EVs and to develop effective methods to control infections and contaminations of cell cultures by mycoplasmas. In the present study, we have documented for the first time the proteins in EVs secreted by mycoplasma vesicular proteins identified in this study are likely involved in the adaptation of bacteria to stressors, survival in microbial communities and pathogen-host interactions. These findings suggest that the secretion of EVs is an evolutionally conserved and universal process that occurs in organisms from the simplest wall-less bacteria to complex organisms and indicate the necessity of developing new approaches to control infects. (hide) EV-METRIC 44% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Mycoplasma Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.1 µm filter + dUC + Iodixanol-DG + UF Adj. k-factor 95.8 (pelleting) Protein markers EV: HSP20 non-EV: None Proteomics yes Show all info Study aim Omics Sample Species Acholeplasma laidlawii Sample Type Mycoplasma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type MLA80 Pelleting: adjusted k-factor 95.8 Density gradient Density medium Iodixanol Lowest density fraction 10 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Filtration steps 0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Detected EV-associated proteins HSP20 ELISA Detected EV-associated proteins HSP20 Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM/ immune EM/ scanning EM/ atomic force EM Image type Close-up, Wide-field

	EV140238	3/3	Mus musculus	Cell culture supernatant	dUC Sucrose-DG	Yuyama K	2014	44%
Study summary Full title Decreased amyloid-beta pathologies by intracerebral loading of glycosphingolipid-enriched exosomes in Alzheimer model mice All authors Yuyama K, Sun H, Sakai S, Mitsutake S, Okada M, Tahara H, Furukawa J, Fujitani N, Shinohara Y, Igarashi Y Journal J Biol Chem Abstract Elevated levels of amyloid-? peptide (A?) in the human brain are linked to the pathogenesis of Alzhe (show more...)Elevated levels of amyloid-? peptide (A?) in the human brain are linked to the pathogenesis of Alzheimer disease. Recent in vitro studies have demonstrated that extracellular A? can bind to exosomes, which are cell-secreted nanovesicles with lipid membranes that are known to transport their cargos intercellularly. Such findings suggest that the exosomes are involved in A? metabolism in brain. Here, we found that neuroblastoma-derived exosomes exogenously injected into mouse brains trapped A? and with the associated A? were internalized into brain-resident phagocyte microglia. Accordingly, continuous intracerebral administration of the exosomes into amyloid-? precursor protein transgenic mice resulted in marked reductions in A? levels, amyloid depositions, and A?-mediated synaptotoxicity in the hippocampus. In addition, we determined that glycosphingolipids (GSLs), a group of membrane glycolipids, are highly abundant in the exosomes, and the enriched glycans of the GSLs are essential for A? binding and assembly on the exosomes both in vitro and in vivo. Our data demonstrate that intracerebrally administered exosomes can act as potent scavengers for A? by carrying it on the exosome surface GSLs and suggest a role of exosomes in A? clearance in the central nervous system. Improving A? clearance by exosome administration would provide a novel therapeutic intervention for Alzheimer disease. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG Protein markers EV: Alix/ Flotilin1/ GM1/ Beta-amyloid/ Actin non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.12-1.16 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Density gradient Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.2999999999999998 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ Flotilin1/ GM1/ Beta-amyloid/ Actin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins GM1/ Beta-amyloid/ Actin Fluorescent NTA Characterization: Particle analysis

	EV140044	2/2	Rattus norvegicus/rattus	Cell culture supernatant	0.2 µm filter dUC	Wang X	2014	44%
Study summary Full title Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells All authors Wang X, Huang W, Liu G, Cai W, Millard RW, Wang Y, Chang J, Peng T, Fan GC Journal J Mol Cell Cardiol Abstract Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play crit (show more...)Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Protein markers EV: CD63/ CD81/ AChE non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ AChE ELISA Detected EV-associated proteins AChE Fluorescent NTA Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

	EV140043	1/3	Homo sapiens	Cell culture supernatant	0.45 µm filter dUC	Vargas A	2014	44%
Study summary Full title Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia All authors Vargas A, Zhou S, Éthier-Chiasson M, Flipo D, Lafond J, Gilbert C, Barbeau B Journal FASEB J Abstract Exosomes are extracellular vesicles that mediate intercellular communication and are involved in sev (show more...)Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.45 µm filter + dUC Protein markers EV: CD63/ TSG101/ AChE non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Wash: volume per pellet (ml) 10 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ TSG101/ AChE Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins AChE Fluorescent NTA Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140041	1/1	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG (valid.)	Toda Y	2014	44%
Study summary Full title Effective internalization of U251-MG-secreted exosomes into cancer cells and characterization of their lipid components All authors Toda Y, Takata K, Nakagawa Y, Kawakami H, Fujioka S, Kobayashi K, Hattori Y, Kitamura Y, Akaji K, Ashihara E Journal Biochem Biophys Res Commun Abstract Exosomes, the natural vehicles of various biological molecules, have been examined in several resear (show more...)Exosomes, the natural vehicles of various biological molecules, have been examined in several research fields including drug delivery. Although understanding of the biological functions of exosomes has increased, how exosomes are transported between cells remains unclear. We hypothesized that cell tropism is important for effective exosomal intercellular communication and that parental cells regulate exosome movement by modulating constituent exosomal molecules. Herein, we demonstrated the strong translocation of glioblastoma-derived exosomes (U251exo) into their parental (U251) cells, breast cancer (MDA-MB-231) cells, and fibrosarcoma (HT-1080). Furthermore, disruption of proteins of U251exo by enzymatic treatment did not affect their uptake. Therefore, we focused on lipid molecules of U251exo with the expectation that they are crucial for effective incorporation of U251exo by cancer cells. Phosphatidylethanolamine was identified as a unique lipid component of U251-MG cell-derived extracellular vesicles. From these results, valuable insight is provided into the targeting of U251exo to cancer cells, which will help to develop a cancer-targeted drug delivery system. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: CD63/ TSG101 non-EV: None Proteomics no EV density (g/ml) 1.160 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ TSG101 Fluorescent NTA Characterization: Particle analysis DLS EM EM-type atomic force EM Image type Close-up, Wide-field

	EV140099	1/1	Homo sapiens	Cell culture supernatant	dUC UF	Sinha A	2014	44%
Study summary Full title In-depth proteomic analyses of ovarian cancer cell line exosomes reveals differential enrichment of functional categories compared to the NCI 60 proteome All authors Sinha A, Ignatchenko V, Ignatchenko A, Mejia-Guerrero S, Kislinger T Journal Biochem Biophys Res Commun Abstract Molecular communication between cancer cells and its stromal microenvironment is a key factor for ca (show more...)Molecular communication between cancer cells and its stromal microenvironment is a key factor for cancer progression. Alongside classic secretory pathways, it has recently been proposed that small membranous vesicles are alternative mediators of intercellular communication. Exosomes carry an effector-rich proteome with the ability to modulate various functional properties of the recipient cell. In this study, exosomes isolated from four epithelial ovarian cancer cell lines (OVCAR3, OVCAR433, OVCAR5 and SKOV3) were characterized using mass spectrometry-based proteomics. Using an optimized workflow consisting of efficient exosome solubilization and the latest generation of proteomic instrumentation, we demonstrate improved detection depth. Systematic comparison of our cancer cell line exosome proteome against public data (Exocarta) and the recently published NCI 60 proteome revealed enrichment of functional categories related to signaling biology and biomarker discovery. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + UF Adj. k-factor 101.8 (pelleting) / 101.8 (washing) Protein markers EV: CD81/ Flotilin1 non-EV: Cell organelle protein/ "VDAC1/ Calreticulin/ Aconitase 2/ GAPDH/ Beta-actin" Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 101.8 Wash: volume per pellet (ml) 9 Wash: Rotor Type 70.1Ti Wash: adjusted k-factor 101.8 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Flotilin1 Detected contaminants Cell organelle protein/ "VDAC1/ Calreticulin/ Aconitase 2/ GAPDH/ Beta-actin" Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140011	1/2	Homo sapiens	BALF	0.2 µm filter dUC	Rodríguez M	2014	44%
Study summary Full title Different exosome cargo from plasma/bronchoalveolar lavage in non-small-cell lung cancer All authors Rodríguez M, Silva J, López-Alfonso A, López-Muñiz MB, Peña C, Domínguez G, García JM, López-Gónzalez A, Méndez M, Provencio M, García V, Bonilla F Journal Genes Chromosomes Cancer Abstract Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth, metastasis, development o (show more...)Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth, metastasis, development of drug resistance, and immunosuppression. However, little is known about the exosomes isolated from bronchoalveolar lavage (BAL) in patients with lung neoplasm. Exosomes isolated in plasma and BAL from 30 and 75 patients with tumor and nontumor pathology were quantified by acetylcholinesterase activity and characterized by Western Blot, Electron Microscopy, and Nanoparticle Tracking Analysis. Differences in exosome cargo were analyzed by miRNA quantitative PCR in pooled samples and validated in a second series of patients. More exosomes were detected in plasma than in BAL in both groups (P < 0.001). The most miRNAs evaluated by PCR array were detected in tumor plasma, tumor BAL, and nontumor BAL pools, but only 56% were detected in the nontumor plasma pool. Comparing the top miRNAs with the highest levels detected in each pool, we found close homology only between the BAL samples of the two pathologies. In tumor plasma, we found a higher percentage of miRNAs with increased levels than in tumor BAL or in nontumor plasma. The data reveal differences between BAL and plasma exosome amount and miRNA content. (hide) EV-METRIC 44% (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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type BALF Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Protein markers EV: CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type BALF Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140011	2/2	Homo sapiens	Blood plasma	0.2 µm filter dUC	Rodríguez M	2014	44%
Study summary Full title Different exosome cargo from plasma/bronchoalveolar lavage in non-small-cell lung cancer All authors Rodríguez M, Silva J, López-Alfonso A, López-Muñiz MB, Peña C, Domínguez G, García JM, López-Gónzalez A, Méndez M, Provencio M, García V, Bonilla F Journal Genes Chromosomes Cancer Abstract Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth, metastasis, development o (show more...)Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth, metastasis, development of drug resistance, and immunosuppression. However, little is known about the exosomes isolated from bronchoalveolar lavage (BAL) in patients with lung neoplasm. Exosomes isolated in plasma and BAL from 30 and 75 patients with tumor and nontumor pathology were quantified by acetylcholinesterase activity and characterized by Western Blot, Electron Microscopy, and Nanoparticle Tracking Analysis. Differences in exosome cargo were analyzed by miRNA quantitative PCR in pooled samples and validated in a second series of patients. More exosomes were detected in plasma than in BAL in both groups (P < 0.001). The most miRNAs evaluated by PCR array were detected in tumor plasma, tumor BAL, and nontumor BAL pools, but only 56% were detected in the nontumor plasma pool. Comparing the top miRNAs with the highest levels detected in each pool, we found close homology only between the BAL samples of the two pathologies. In tumor plasma, we found a higher percentage of miRNAs with increased levels than in tumor BAL or in nontumor plasma. The data reveal differences between BAL and plasma exosome amount and miRNA content. (hide) EV-METRIC 44% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Protein markers EV: CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140092	1/2	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG (valid.)	Putz U	2014	44%
Study summary Full title PTEN secretion in exosomes All authors Putz U, Mah S, Goh CP, Low LH, Howitt J, Tan SS Journal Methods Abstract PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but th (show more...)PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but the concept that it can be secreted and taken up by recipient cells is revolutionary. Since then, various laboratories have reported that PTEN is indeed secreted and available for uptake by other cells in at least two different guises. First, PTEN may be packaged and exported within extracellular vesicles (EV) called exosomes. Second, PTEN may also be secreted as a naked protein in a longer isoform called PTEN-long. While the conditions favouring the secretion of PTEN-long remain unknown, PTEN secretion in exosomes is enhanced by the Ndfip1/Nedd4 ubiquitination system. In this report, we describe conditions for packaging PTEN in exosomes and their potential use for mediating non cell-autonomous functions in recipient cells. We suggest that this mode of PTEN transfer may potentially provide beneficial PTEN for tumor suppression, however it may also propagate deleterious versions of mutated PTEN causing tumorigenesis. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ TSG101/ Flotillin non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.140 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 10 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ TSG101/ Flotillin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Flotillin Fluorescent NTA Characterization: Particle analysis EM EM-type immune EM Image type Close-up

	EV140092	2/2	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG (valid.)	Putz U	2014	44%
Study summary Full title PTEN secretion in exosomes All authors Putz U, Mah S, Goh CP, Low LH, Howitt J, Tan SS Journal Methods Abstract PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but th (show more...)PTEN was discovered as a membrane-associated tumor suppressor protein nearly two decades ago, but the concept that it can be secreted and taken up by recipient cells is revolutionary. Since then, various laboratories have reported that PTEN is indeed secreted and available for uptake by other cells in at least two different guises. First, PTEN may be packaged and exported within extracellular vesicles (EV) called exosomes. Second, PTEN may also be secreted as a naked protein in a longer isoform called PTEN-long. While the conditions favouring the secretion of PTEN-long remain unknown, PTEN secretion in exosomes is enhanced by the Ndfip1/Nedd4 ubiquitination system. In this report, we describe conditions for packaging PTEN in exosomes and their potential use for mediating non cell-autonomous functions in recipient cells. We suggest that this mode of PTEN transfer may potentially provide beneficial PTEN for tumor suppression, however it may also propagate deleterious versions of mutated PTEN causing tumorigenesis. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ TSG101/ Flotillin non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.140 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 10 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ TSG101/ Flotillin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Flotillin Fluorescent NTA Characterization: Particle analysis EM EM-type immune EM Image type Close-up

	EV140131	2/3	Homo sapiens	Platelet supernatant	dUC filter Iodixanol-DG	Pienimaeki-Roemer A	2014	44%
Study summary Full title Lipidomic and proteomic characterization of platelet extracellular vesicle subfractions from senescent platelets All authors Pienimaeki-Roemer A, Kuhlmann K, Böttcher A, Konovalova T, Black A, Orsó E, Liebisch G, Ahrens M, Eisenacher M, Meyer HE, Schmitz G Journal Transfusion Abstract BACKGROUND: Platelets (PLTs) in stored PLT concentrates (PLCs) release PLT extracellular vesicles (P (show more...)BACKGROUND: Platelets (PLTs) in stored PLT concentrates (PLCs) release PLT extracellular vesicles (PL-EVs) induced by senescence and activation, resembling the PLT storage lesion. No comprehensive classification or molecular characterization of senescence-induced PL-EVs exists to understand PL-EV heterogeneity. STUDY DESIGN AND METHODS: PL-EVs from 5-day-stored PLCs from healthy individuals were isolated and subfractionated by differential centrifugation, filtration, and density gradient ultracentrifugation into five PLT microvesicle (PL-MV) subfractions (Fraction [F]1-F5) and PLT exosomes (PL-EXs). PL-EV size, concentration, and composition were analyzed by nanoparticle tracking analysis, flow cytometry, and lipid and protein mass spectrometry. Protein data were verified by Western blot. RESULTS: PL-EVs showed overlapping mean particle sizes of 180 to 260 nm, but differed significantly in composition. Less dense, intermediate, and dense PL-MVs enriched specific lipidomic and proteomic markers related to the plasma membrane, intracellular membranes, PLT granules, mitochondria, and PLT activation. ?-Synuclein (81% of total) accumulated in F1 and F2, amyloid-? (A?) precursor protein in F3 and F4 (84%), and apolipoprotein (Apo)E (88%) and ApoJ (92%) in F3 to F5. PL-EXs enriched lipid species and proteins, with high abundance of lipid raft, PLT adhesion, and immune response-related markers. CONCLUSION: Differential lipid and protein compositions of PL-EVs suggest their unique cellular origins and functions, partly overlapping with PLT granule secretion. Dense PL-MVs might represent autophagic vesicles released during PLT activation and apoptosis and PL-EXs resemble lipid rafts, with a potential role in PLT aggregation and immunity. Segregation of ?-synuclein and A? precursor protein, ApoE, and ApoJ into less dense and dense PL-MVs, respectively, show their differential carrier role of neurologic disease-related cargo. (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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Platelet supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + filter + Iodixanol-DG Adj. k-factor 784.6 (pelleting) / 784.6 (washing) Protein markers EV: Alix/ CD63/ CD9 non-EV: None Proteomics yes EV density (g/ml) 1.12-1.15 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Platelet supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting: time(min) 40 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 784.6 Wash: Rotor Type 70Ti Wash: adjusted k-factor 784.6 Density gradient Density medium Iodixanol Lowest density fraction 10 Highest density fraction 30 Orientation Top-down Rotor type SW41 Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 2 Other Name other isolation method filter Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ CD9 Fluorescent NTA Characterization: Particle analysis NTA

	EV140131	3/3	Homo sapiens	Platelet supernatant	dUC filter Iodixanol-DG	Pienimaeki-Roemer A	2014	44%
Study summary Full title Lipidomic and proteomic characterization of platelet extracellular vesicle subfractions from senescent platelets All authors Pienimaeki-Roemer A, Kuhlmann K, Böttcher A, Konovalova T, Black A, Orsó E, Liebisch G, Ahrens M, Eisenacher M, Meyer HE, Schmitz G Journal Transfusion Abstract BACKGROUND: Platelets (PLTs) in stored PLT concentrates (PLCs) release PLT extracellular vesicles (P (show more...)BACKGROUND: Platelets (PLTs) in stored PLT concentrates (PLCs) release PLT extracellular vesicles (PL-EVs) induced by senescence and activation, resembling the PLT storage lesion. No comprehensive classification or molecular characterization of senescence-induced PL-EVs exists to understand PL-EV heterogeneity. STUDY DESIGN AND METHODS: PL-EVs from 5-day-stored PLCs from healthy individuals were isolated and subfractionated by differential centrifugation, filtration, and density gradient ultracentrifugation into five PLT microvesicle (PL-MV) subfractions (Fraction [F]1-F5) and PLT exosomes (PL-EXs). PL-EV size, concentration, and composition were analyzed by nanoparticle tracking analysis, flow cytometry, and lipid and protein mass spectrometry. Protein data were verified by Western blot. RESULTS: PL-EVs showed overlapping mean particle sizes of 180 to 260 nm, but differed significantly in composition. Less dense, intermediate, and dense PL-MVs enriched specific lipidomic and proteomic markers related to the plasma membrane, intracellular membranes, PLT granules, mitochondria, and PLT activation. ?-Synuclein (81% of total) accumulated in F1 and F2, amyloid-? (A?) precursor protein in F3 and F4 (84%), and apolipoprotein (Apo)E (88%) and ApoJ (92%) in F3 to F5. PL-EXs enriched lipid species and proteins, with high abundance of lipid raft, PLT adhesion, and immune response-related markers. CONCLUSION: Differential lipid and protein compositions of PL-EVs suggest their unique cellular origins and functions, partly overlapping with PLT granule secretion. Dense PL-MVs might represent autophagic vesicles released during PLT activation and apoptosis and PL-EXs resemble lipid rafts, with a potential role in PLT aggregation and immunity. Segregation of ?-synuclein and A? precursor protein, ApoE, and ApoJ into less dense and dense PL-MVs, respectively, show their differential carrier role of neurologic disease-related cargo. (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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Platelet supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + filter + Iodixanol-DG Adj. k-factor 130.7 (pelleting) / 130.7 (washing) Protein markers EV: Alix/ CD63/ CD9 non-EV: None Proteomics yes EV density (g/ml) 1.12-1.15 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Platelet supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 130.7 Wash: Rotor Type 70Ti Wash: adjusted k-factor 130.7 Density gradient Density medium Iodixanol Lowest density fraction 10 Highest density fraction 30 Orientation Top-down Rotor type SW41 Speed (g) 100000 Pelleting-wash: volume per pellet (mL) 2 Other Name other isolation method filter Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ CD9 Fluorescent NTA Characterization: Particle analysis NTA

	EV140038	1/1	Homo sapiens	Cell culture supernatant	dUC UF	Park S	2014	44%
Study summary Full title Neuroblastoma SH-SY5Y cell-derived exosomes stimulate dendrite-like outgrowths and modify the differentiation of A375 melanoma cells All authors Park S, Ahn ES, Kim Y Journal Cell Biol Int Abstract The identification of small vesicles released by many cell types as tools of intercellular communica (show more...)The identification of small vesicles released by many cell types as tools of intercellular communication is proposed. Here, we identify SH-SY5Y neuroblastoma-derived exosomes comprised of major histocompatibility complex II (MHC II), Hsp90 and flotillin-1. Our data also suggest that, when applied extracellularly, exosomes released from neuronal cells stimulate dendrite-like outgrowth and melanogenesis of A375 melanoma cells through the mitogen-activated protein kinase (MAP kinase), extracellular signal-regulated kinase 1 (ERK1) activation. These results suggest a modification of differentiation of melanocyte by the treatment of neuronal cell exosomes. Since exosomes from neuronal cells have the capacity to affect melanoma cells, they could be generally implicated in intercellular communication between different types of cells. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + UF Adj. k-factor 156.9 (pelleting) / 126 (washing) Protein markers EV: Flotilin1/ HSP90/ MHC2 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: Rotor Type 90Ti Wash: adjusted k-factor 126.0 Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ HSP90/ MHC2 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins MHC2 Fluorescent NTA Characterization: Particle analysis DLS EM EM-type cryo EM Image type Wide-field

	EV140203	1/1	Mus musculus	Cell culture supernatant	0.2 µm filter dUC Iodixanol-DG (valid.)	Padro CJ	2014	44%
Study summary Full title Adrenergic regulation of IgE involves modulation of CD23 and ADAM10 expression on exosomes All authors Padro CJ, Shawler TM, Gormley MG, Sanders VM Journal J Immunol Abstract Soluble CD23 plays a role in the positive regulation of an IgE response. Engagement of the ?2 adrene (show more...)Soluble CD23 plays a role in the positive regulation of an IgE response. Engagement of the ?2 adrenergic receptor (?2AR) on a B cell is known to enhance the level of both soluble CD23 and IgE, although the mechanism by which this occurs is not completely understood. In this study, we report that, in comparison with a CD40 ligand/IL-4-primed murine B cell alone, ?2AR engagement on a primed B cell increased gene expression of a disintegrin and metalloproteinase (ADAM)10, which is the primary sheddase of CD23, as well as protein expression of both CD23 and ADAM10, in a protein kinase A- and p38 MAPK-dependent manner, and promoted the localization of these proteins to exosomes as early as 2 d after priming, as determined by both Western blot and flow cytometry and confirmed by electron microscopy. In comparison with isolated exosomes released from primed B cells alone, the transfer of exosomes released from ?2AR agonist-exposed primed B cells to cultures of recipient primed B cells resulted in an increase in the level of IgE produced per cell, without affecting the number of cells producing IgE, as determined by ELISPOT. These effects still occurred when a ?2AR antagonist was added along with the transfer to block residual agonist, and they failed to occur when exosomes were isolated from ?2AR-deficient B cells. These findings suggest that the mechanism responsible for mediating the ?2AR-induced increase in IgE involves a shuttling of the ?2AR-induced increase in CD23 and ADAM10 proteins to exosomes that subsequently mediate an increase in IgE. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Iodixanol-DG (valid.) Adj. k-factor 255.8 (pelleting) Protein markers EV: CD63/ ADAM10 non-EV: None Proteomics no EV density (g/ml) 1.05-1.1 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 1.00g/L Highest density fraction 1.25g/L Orientation Top-down Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ ADAM10 ELISA Detected EV-associated proteins ADAM10 Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140088	1/1	Homo sapiens	Cell culture supernatant	0.1 µm filter dUC UF	Ohshima K	2014	44%
Study summary Full title Exosome-mediated extracellular release of polyadenylate-binding protein 1 in human metastatic duodenal cancer cells All authors Ohshima K, Kanto K, Hatakeyama K, Ide T, Wakabayashi-Nakao K, Watanabe Y, Sakura N, Terashima M, Yamaguchi K, Mochizuki T Journal Proteomics Abstract Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidi (show more...)Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis- and endocytosis-like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ-521) and metastatic (AZ-P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC-MS/MS and Western blot analyses, polyadenylate-binding protein 1 (PABP1) was found to be predominantly abundant in AZ-P7a exosomes. The amount of exosomal PABP1 in AZ-P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin-proteasome pathway (MG-132 and PYR-41). Treatment of AZ-P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1-immunoreactive products cleaved via a proteolysis-like process. Taken together, these results suggest that AZ-P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome-mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.1 µm filter + dUC + UF Adj. k-factor 156.9 (pelleting) / 93.46 (washing) Protein markers EV: Alix/ Syntenin/ Beta-actin/ GAPDH/ Tubulin non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 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 Detected EV-associated proteins Alix/ Syntenin/ Beta-actin/ GAPDH/ Tubulin ELISA Detected EV-associated proteins Beta-actin/ GAPDH/ Tubulin Fluorescent NTA Characterization: Particle analysis

	EV140119	2/3	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC Iodixanol-DG (valid.)	Liu Z	2014	44%
Study summary Full title Exosome-associated hepatitis C virus in cell cultures and patient plasma All authors Liu Z, Zhang X, Yu Q, He JJ Journal Biochem Biophys Res Commun Abstract Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-c (show more...)Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Iodixanol-DG (valid.) Adj. k-factor 55.47 (pelleting) Protein markers EV: CD63/ HSP70/ AChE non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting: time(min) 90 Pelleting: rotor type SW55 Pelleting: adjusted k-factor 55.47 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 6 Highest density fraction 24 Orientation Top-down Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ HSP70/ AChE ELISA Detected EV-associated proteins AChE Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140119	3/3	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC Iodixanol-DG (valid.)	Liu Z	2014	44%
Study summary Full title Exosome-associated hepatitis C virus in cell cultures and patient plasma All authors Liu Z, Zhang X, Yu Q, He JJ Journal Biochem Biophys Res Commun Abstract Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-c (show more...)Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Iodixanol-DG (valid.) Adj. k-factor 195.3 (pelleting) Protein markers EV: CD63/ HSP70/ AChE non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 195.3 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 6 Highest density fraction 24 Orientation Top-down Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ HSP70/ AChE ELISA Detected EV-associated proteins AChE Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140031	1/2	Homo sapiens	Urine	dUC UF	Kosanovi? M	2014	44%
Study summary Full title Isolation of urinary extracellular vesicles from Tamm- Horsfall protein-depleted urine and their application in the development of a lectin-exosome-binding assay All authors Kosanovic M, Jankovic M Journal Biotechniques Abstract Urine is a readily available source of relatively large quantities of extracellular vesicles (EVs). (show more...)Urine is a readily available source of relatively large quantities of extracellular vesicles (EVs). However, the isolation of urinary EVs (uEVs) is complicated by the presence of Tamm-Horsfall protein (THP), which polymerizes and co-precipitates as a contaminant. This may make glycan analysis of uEVs difficult since THP is heavily glycosylated. To facilitate glycosylation analysis and address the need for elimination of non-uEV glycans, we present a modification of the uEV isolation procedure and use the isolated uEVs in the development of a lectin-exosome binding assay. Salt precipitation was employed to remove THP under conditions originally described for its separation from urine, followed by differential centrifugation. The quality of the isolated uEVs was examined by electron microscopy, SDS-PAGE, and immunoblotting. The uEVs were subsequently immobilized on solid phase and probed with labeled plant lectins using the lectin-exosome binding assay. Our results indicate that the isolated uEVs had preserved structural integrity and reacted with labeled plant lectins in a selective, carbohydrate-dependent manner. The basic lectin binding pattern of uEVs obtained by our method can be used as a reference for assessing the composition of their surface glycans in different physiological and pathological conditions. (hide) EV-METRIC 44% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles exosomes / extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + UF Adj. k-factor 157.1 (pelleting) Protein markers EV: CD63/ Galectin3 non-EV: Tamm-Horsfall glycoprotein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 6 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Galectin3 Detected contaminants Tamm-Horsfall glycoprotein ELISA Detected EV-associated proteins CD63/ Galectin3 Detected contaminants Tamm-Horsfall glycoprotein Fluorescent NTA Characterization: Particle analysis

	EV140031	2/2	Homo sapiens	Urine	0.2 µm filter dUC UF	Kosanovi? M	2014	44%
Study summary Full title Isolation of urinary extracellular vesicles from Tamm- Horsfall protein-depleted urine and their application in the development of a lectin-exosome-binding assay All authors Kosanovic M, Jankovic M Journal Biotechniques Abstract Urine is a readily available source of relatively large quantities of extracellular vesicles (EVs). (show more...)Urine is a readily available source of relatively large quantities of extracellular vesicles (EVs). However, the isolation of urinary EVs (uEVs) is complicated by the presence of Tamm-Horsfall protein (THP), which polymerizes and co-precipitates as a contaminant. This may make glycan analysis of uEVs difficult since THP is heavily glycosylated. To facilitate glycosylation analysis and address the need for elimination of non-uEV glycans, we present a modification of the uEV isolation procedure and use the isolated uEVs in the development of a lectin-exosome binding assay. Salt precipitation was employed to remove THP under conditions originally described for its separation from urine, followed by differential centrifugation. The quality of the isolated uEVs was examined by electron microscopy, SDS-PAGE, and immunoblotting. The uEVs were subsequently immobilized on solid phase and probed with labeled plant lectins using the lectin-exosome binding assay. Our results indicate that the isolated uEVs had preserved structural integrity and reacted with labeled plant lectins in a selective, carbohydrate-dependent manner. The basic lectin binding pattern of uEVs obtained by our method can be used as a reference for assessing the composition of their surface glycans in different physiological and pathological conditions. (hide) EV-METRIC 44% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles exosomes / extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + UF Adj. k-factor 157.1 (pelleting) Protein markers EV: CD63/ Galectin3 non-EV: Tamm-Horsfall glycoprotein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Galectin3 Detected contaminants Tamm-Horsfall glycoprotein ELISA Detected EV-associated proteins CD63/ Galectin3 Detected contaminants Tamm-Horsfall glycoprotein Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM/ scanning EM Image type Wide-field

	EV140083	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC Sucrose-DG (valid.)	Koch R	2014	44%
Study summary Full title Populational equilibrium through exosome-mediated Wnt signaling in tumor progression of diffuse large B-cell lymphoma All authors Koch R, Demant M, Aung T, Diering N, Cicholas A, Chapuy B, Wenzel D, Lahmann M, Güntsch A, Kiecke C, Becker S, Hupfeld T, Venkataramani V, Ziepert M, Opitz L, Klapper W, Trümper L, Wulf GG Journal Blood Abstract Tumors are composed of phenotypically heterogeneous cell populations. The nongenomic mechanisms unde (show more...)Tumors are composed of phenotypically heterogeneous cell populations. The nongenomic mechanisms underlying transitions and interactions between cell populations are largely unknown. Here, we show that diffuse large B-cell lymphomas possess a self-organized infrastructure comprising side population (SP) and non-SP cells, where transitions between clonogenic states are modulated by exosome-mediated Wnt signaling. DNA methylation modulated SP-non-SP transitions and was correlated with the reciprocal expressions of Wnt signaling pathway agonist Wnt3a in SP cells and the antagonist secreted frizzled-related protein 4 in non-SP cells. Lymphoma SP cells exhibited autonomous clonogenicity and exported Wnt3a via exosomes to neighboring cells, thus modulating population equilibrium in the tumor. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Adj. k-factor 213.3 (pelleting) Protein markers EV: Flotillin2 non-EV: None Proteomics no EV density (g/ml) 1.160 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 240 Pelleting: rotor type 32Ti Pelleting: adjusted k-factor 213.3 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.25 Orientation Top-down Rotor type SW32 Speed (g) 110000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotillin2 ELISA Detected EV-associated proteins Flotillin2 Fluorescent NTA Characterization: Particle analysis EM EM-type immune EM Image type Close-up, Wide-field

	EV140179	1/1	Homo sapiens	Cell culture supernatant	dUC Iodixanol-DG (valid.) UF	Klinker MW	2014	44%
Study summary Full title Human B Cell-Derived Lymphoblastoid Cell Lines Constitutively Produce Fas Ligand and Secrete MHCII(+)FasL(+) Killer Exosomes All authors Klinker MW, Lizzio V, Reed TJ, Fox DA, Lundy SK Journal Front Immunol Abstract Immune suppression mediated by exosomes is an emerging concept with potentially immense utility for (show more...)Immune suppression mediated by exosomes is an emerging concept with potentially immense utility for immunotherapy in a variety of inflammatory contexts, including allogeneic transplantation. Exosomes containing the apoptosis-inducing molecule Fas ligand (FasL) have demonstrated efficacy in inhibiting antigen-specific immune responses upon adoptive transfer in animal models. We report here that a very high frequency of human B cell-derived lymphoblastoid cell lines (LCL) constitutively produce MHCII(+)FasL(+) exosomes that can induce apoptosis in CD4(+) T cells. All LCL tested for this study (>20 independent cell lines) showed robust expression of FasL, but had no detectable FasL on the cell surface. Given this intracellular sequestration, we hypothesized that FasL in LCL was retained in the secretory lysosome and secreted via exosomes. Indeed, we found both MHCII and FasL proteins present in LCL-derived exosomes, and using a bead-based exosome capture assay demonstrated the presence of MHCII(+)FasL(+) exosomes among those secreted by LCL. Using two independent experimental approaches, we demonstrated that LCL-derived exosomes were capable of inducing antigen-specific apoptosis in autologous CD4(+) T cells. These results suggest that LCL-derived exosomes may present a realistic source of immunosuppressive exosomes that could reduce or eliminate T cell-mediated responses against donor-derived antigens in transplant recipients. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG (valid.) + UF Protein markers EV: MHC2/ FasL/ MHC2/ Beta-actin non-EV: FasL Proteomics no EV density (g/ml) 1.160 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60-240 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 1.03g/l Highest density fraction 1.27g/l Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins MHC2/ FasL/ MHC2/ Beta-actin Detected contaminants FasL ELISA Detected EV-associated proteins MHC2/ FasL/ MHC2/ Beta-actin Flow cytometry specific beads Selected surface protein(s) Yes Fluorescent NTA Characterization: Particle analysis Particle analysis: flow cytometry

	EV140008	1/2	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	Kahlert C	2014	44%
Study summary Full title Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer All authors Kahlert C, Melo SA, Protopopov A, Tang J, Seth S, Koch M, Zhang J, Weitz J, Chin L, Futreal A, Kalluri R Journal J Biol Chem Abstract Exosomes are small vesicles (50-150 nm) of endocytic origin that are released by many different cell (show more...)Exosomes are small vesicles (50-150 nm) of endocytic origin that are released by many different cell types. Exosomes in the tumor microenvironment may play a key role in facilitating cell-cell communication. Exosomes are reported to predominantly contain RNA and proteins. In this study, we investigated whether exosomes from pancreatic cancer cells and serum from patients with pancreatic ductal adenocarcinoma contain genomic DNA. Our results provide evidence that exosomes contain >10-kb fragments of double-stranded genomic DNA. Mutations in KRAS and p53 can be detected using genomic DNA from exosomes derived from pancreatic cancer cell lines and serum from patients with pancreatic cancer. In addition, using whole genome sequencing, we demonstrate that serum exosomes from patients with pancreatic cancer contain genomic DNA spanning all chromosomes. These results indicate that serum-derived exosomes can be used to determine genomic DNA mutations for cancer prediction, treatment, and therapy resistance. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Protein markers EV: CD63/ CD9/ TSG101 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting: time(min) 120 Wash: volume per pellet (ml) 30 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD9/ TSG101 Fluorescent NTA Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV140008	2/2	Homo sapiens	Serum	0.2 µm filter dUC UF	Kahlert C	2014	44%
Study summary Full title Identification of double-stranded genomic DNA spanning all chromosomes with mutated KRAS and p53 DNA in the serum exosomes of patients with pancreatic cancer All authors Kahlert C, Melo SA, Protopopov A, Tang J, Seth S, Koch M, Zhang J, Weitz J, Chin L, Futreal A, Kalluri R Journal J Biol Chem Abstract Exosomes are small vesicles (50-150 nm) of endocytic origin that are released by many different cell (show more...)Exosomes are small vesicles (50-150 nm) of endocytic origin that are released by many different cell types. Exosomes in the tumor microenvironment may play a key role in facilitating cell-cell communication. Exosomes are reported to predominantly contain RNA and proteins. In this study, we investigated whether exosomes from pancreatic cancer cells and serum from patients with pancreatic ductal adenocarcinoma contain genomic DNA. Our results provide evidence that exosomes contain >10-kb fragments of double-stranded genomic DNA. Mutations in KRAS and p53 can be detected using genomic DNA from exosomes derived from pancreatic cancer cell lines and serum from patients with pancreatic cancer. In addition, using whole genome sequencing, we demonstrate that serum exosomes from patients with pancreatic cancer contain genomic DNA spanning all chromosomes. These results indicate that serum-derived exosomes can be used to determine genomic DNA mutations for cancer prediction, treatment, and therapy resistance. (hide) EV-METRIC 44% (95th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + UF Protein markers EV: CD63/ CD9/ TSG101 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Isolation Method Differential ultracentrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting: time(min) 960 Wash: volume per pellet (ml) 11 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD9/ TSG101 Fluorescent NTA Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV140007	1/1	Mus musculus	Cell culture supernatant	dUC Iodixanol-DG (valid.)	Ju R	2014	44%
Study summary Full title Angiopoietin-2 secretion by endothelial cell exosomes: regulation by the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) and syndecan-4/syntenin pathways All authors Ju R, Zhuang ZW, Zhang J, Lanahan AA, Kyriakides T, Sessa WC, Simons M Journal J Biol Chem Abstract Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor (show more...)Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG (valid.) Protein markers EV: CD63/ HSP90/ Syntenin non-EV: Cell organelle protein Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Bottom-up Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ HSP90/ Syntenin Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140030	2/2	Mus musculus	Cell culture supernatant	dUC Microfluidics UF	Jo W	2014	44%
Study summary Full title Microfluidic fabrication of cell-derived nanovesicles as endogenous RNA carriers All authors Jo W, Jeong D, Kim J, Cho S, Jang SC, Han C, Kang JY, Gho YS, Park J Journal Lab Chip Abstract Exosomes/microvesicles are known to shuttle biological signals between cells, possibly by transferri (show more...)Exosomes/microvesicles are known to shuttle biological signals between cells, possibly by transferring biological signal components such as encapsulated RNAs and proteins, plasma membrane proteins, or both. Therefore exosomes are being considered for use as RNA and protein delivery vehicles for various therapeutic applications. However, living cells in nature secrete only a small number of exosomes, and procedures to collect them are complex; these complications impede their use in mass delivery of components to targeted cells. We propose a novel and efficient method that forces cells through hydrophilic microchannels to generate artificial nanovesicles. These mimetic nanovesicles contain mRNAs, intracellular proteins and plasma membrane proteins, and are shaped like cell-secreted exosomes. When recipient cells are exposed to nanovesicles from embryonic stem cells, mRNAs of Oct 3/4 and Nanog are transferred from embryonic stem cells to the target cells. This result suggests that mimetic nanovesicles can be used as vehicles to deliver RNA. This nanovesicle formation method is expected to be used in exosome research and to have applications in drug and RNA-delivery systems. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles Nano(-sized) vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Microfluidics + UF Protein markers EV: Actin/ Nanog/ ICAM1 non-EV: None Proteomics no Show all info Study aim Other/Generation of nanovesicles Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Other Name other isolation method Microfluidics Characterization: Protein analysis Western Blot Detected EV-associated proteins Actin/ Nanog/ ICAM1 ELISA Detected EV-associated proteins Actin/ Nanog/ ICAM1 Fluorescent NTA Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140006	1/1	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Quantitative proteomics of fractionated membrane and lumen exosome proteins from isogenic metastatic and nonmetastatic bladder cancer cells reveal differential expression of EMT factors All authors Jeppesen DK, Nawrocki A, Jensen SG, Thorsen K, Whitehead B, Howard KA, Dyrskjøt L, Ørntoft TF, Larsen MR, Ostenfeld MS Journal Proteomics Abstract Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into th (show more...)Cancer cells secrete soluble factors and various extracellular vesicles, including exosomes, into their tissue microenvironment. The secretion of exosomes is speculated to facilitate local invasion and metastatic spread. Here, we used an in vivo metastasis model of human bladder carcinoma cell line T24 without metastatic capacity and its two isogenic derivate cell lines SLT4 and FL3, which form metastases in the lungs and liver of mice, respectively. Cultivation in CLAD1000 bioreactors rather than conventional culture flasks resulted in a 13- to 16-fold increased exosome yield and facilitated quantitative proteomics of fractionated exosomes. Exosomes from T24, SLT4, and FL3 cells were partitioned into membrane and luminal fractions and changes in protein abundance related to the gain of metastatic capacity were identified by quantitative iTRAQ proteomics. We identified several proteins linked to epithelial-mesenchymal transition, including increased abundance of vimentin and hepatoma-derived growth factor in the membrane, and casein kinase II ? and annexin A2 in the lumen of exosomes, respectively, from metastatic cells. The change in exosome protein abundance correlated little, although significant for FL3 versus T24, with changes in cellular mRNA expression. Our proteomic approach may help identification of proteins in the membrane and lumen of exosomes potentially involved in the metastatic process. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: Alix/ CD63/ CD81/ CD9/ Syntenin/ Beta-actin/ GAPDH 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 serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ CD81/ CD9/ Syntenin/ Beta-actin/ GAPDH Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Beta-actin/ GAPDH Fluorescent NTA Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140029	1/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 475.5 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 475.5 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	2/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 234.2 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 234.2 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	3/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 156.9 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	4/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 117.9 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 117.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	5/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 93.96 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 93.96 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	6/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 78.45 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	7/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 475.5 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 475.5 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	8/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 234.2 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 234.2 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	9/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 156.9 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	10/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 117.9 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 117.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	11/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 93.96 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 93.96 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140029	12/12	Homo sapiens	Cell culture supernatant	dUC	Jeppesen DK	2014	44%
Study summary Full title Comparative analysis of discrete exosome fractions obtained by differential centrifugation All authors Jeppesen DK, Hvam ML, Primdahl-Bengtson B, Boysen AT, Whitehead B, Dyrskjøt L, Orntoft TF, Howard KA, Ostenfeld MS Journal J Extracell Vesicles Abstract BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ i (show more...)BACKGROUND: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. METHODS: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. RESULTS: NTA revealed the lowest background particle count in Dulbecco's Modified Eagle's Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. CONCLUSIONS: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 78.45 (pelleting) Protein markers EV: CD81/ Syntenin/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Fluorescent NTA Characterization: Particle analysis NTA

	EV140027	1/1	Homo sapiens	Cell culture supernatant	dUC UF	Inder KL	2014	44%
Study summary Full title Cavin-1/PTRF alters prostate cancer cell-derived extracellular vesicle content and internalization to attenuate extracellular vesicle-mediated osteoclastogenesis and osteoblast proliferation All authors Inder KL, Ruelcke JE, Petelin L, Moon H, Choi E, Rae J, Blumenthal A, Hutmacher D, Saunders NA, Stow JL, Parton RG, Hill MM Journal J Extracell Vesicles Abstract BACKGROUND: Tumour-derived extracellular vesicles (EVs) play a role in tumour progression; however, (show more...)BACKGROUND: Tumour-derived extracellular vesicles (EVs) play a role in tumour progression; however, the spectrum of molecular mechanisms regulating EV secretion and cargo selection remain to be fully elucidated. We have reported that cavin-1 expression in prostate cancer PC3 cells reduced the abundance of a subset of EV proteins, concomitant with reduced xenograft tumour growth and metastasis. METHODS: We examined the functional outcomes and mechanisms of cavin-1 expression on PC3-derived EVs (PC3-EVs). RESULTS: PC3-EVs were internalized by osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs) in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. Strikin gly, EVs derived from cavin-1-expressing PC3 cells (cavin-1-PC3-EVs) failed to induce multinucleate osteoblasts or hOB proliferation. Cavin-1 was not detected in EVs, indicating an indirect mechanism of action. EV morphology, size and quantity were also not affected by cavin-1 expression, suggesting that cavin-1 modulated EV cargo recruitment rather than release. While cavin-1-EVs had no osteoclastogenic function, they were internalized by RAW264.7 cells but at a reduced efficiency compared to control EVs. EV surface proteins are required for internalization of PC3-EVs by RAW264.7 cells, as proteinase K treatment abolished uptake of both control and cavin-1-PC3-EVs. Removal of sialic acid modifications by neuraminidase treatment increased the amount of control PC3-EVs internalized by RAW264.7 cells, without affecting cavin-1-PC3-EVs. This suggests that cavin-1 expression altered the glycosylation modifications on PC3-EV surface. Finally, cavin-1 expression did not affect EV in vivo tissue targeting as both control and cavin-1-PC3-EVs were predominantly retained in the lung and bone 24 hours after injection into mice. DISCUSSION: Taken together, our results reveal a novel pathway for EV cargo sorting, and highlight the potential of utilizing cavin-1-mediated pathways to attenuate metastatic prostate cancer. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + UF Protein markers EV: CD63/ Caveolin1/ Cofilin/ EphA2/ 4F2/ CD98 non-EV: Cell organelle protein Proteomics no TEM measurements 86.9+-4.7 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Caveolin1/ Cofilin/ EphA2/ 4F2/ CD98 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Caveolin1/ Cofilin/ EphA2/ 4F2/ CD98 Fluorescent NTA Characterization: Particle analysis EM EM-type transmission EM/ immune EM Image type Close-up, Wide-field

	EV140123	2/2	Homo sapiens	Blood plasma	0.2 µm filter dUC SEC Sucrose-DG	Hong CS	2014	44%
Study summary Full title Plasma exosomes as markers of therapeutic response in patients with acute myeloid leukemia All authors Hong CS, Muller L, Whiteside TL, Boyiadzis M Journal Front Immunol Abstract PURPOSE: Exosomes isolated from the plasma of newly diagnosed acute myeloid leukemia (AML) patients (show more...)PURPOSE: Exosomes isolated from the plasma of newly diagnosed acute myeloid leukemia (AML) patients have elevated protein and transforming growth factor-beta 1 (TGF-?1) contents and inhibit natural killer (NK) cell cytotoxicity (Haematologica 96, p. 1302, 2011). A potential role of exosomes in predicting responses to chemotherapy (CT) was evaluated in AML patients undergoing treatment. EXPERIMENTAL DESIGN: Plasma was obtained from AML patients at diagnosis (n = 16); post-induction CT (n = 9); during consolidation CT (n = 10); in long-term remission (Lt-CR, n = 5); and from healthy volunteers (n = 7). Exosomes were isolated by size-exclusion chromatography and ultracentrifugation. The exosomal protein, soluble TGF?-1 levels (ELISA), and the TGF-?1 profiles (western blots) were compared among patients' cohorts. The results were correlated with the patients' cytogenetic profile, percentage of leukemic blast, and outcome. RESULTS: At diagnosis, protein and TGF-?1 levels were higher (p < 0.009 and p < 0.004) in AML than control exosomes. These values decreased after induction CT (p < 0.05 and p < 0.004), increased during consolidation CT (p < 0.02 and p < 0.005), and normalized in Lt-CR. While TGF-?1 and protein levels tracked one another, TGF-?1 pro-peptide, latency-associated peptide (LAP), or mature TGF-?1 differentially decorated exosomes isolated before, during, and after CT. Only TGF-?1 pro-peptide was seen in exosomes of controls or Lt-CR patients. During consolidation CT, exosomes carried TGF-?1 pro-peptide, LAP, and low levels of mature TGF-?1. NK cell co-incubation with AML exosomes carrying all three TGF-?1 forms induced down-regulation of NKG2D expression. CONCLUSION: Changes in exosomal protein and/or TGF-?1 content may reflect responses to CT. The exosomal profile may suggest the presence of residual disease in patients considered to have achieved complete remission. (hide) EV-METRIC 44% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + SEC + Sucrose-DG Protein markers EV: CD81 non-EV: None Proteomics no EV density (g/ml) 1.16-1.19 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Density gradient Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Top-down Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81 Flow cytometry specific beads Selected surface protein(s) Yes Fluorescent NTA Characterization: Particle analysis NTA Particle analysis: flow cytometry EM EM-type transmission EM Image type Close-up

	EV140117	2/8	Homo sapiens	Blood plasma	dUC	He M	2014	44%
Study summary Full title Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology All authors He M, Crow J, Roth M, Zeng Y, Godwin AK Journal Lab Chip Abstract Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy (show more...)Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy is difficult, costly, and sometimes not even an option. Tumor-derived exosomes have attracted increasing interest in non-invasive cancer diagnosis and monitoring of treatment response. However, the biology and clinical value of exosomes remains largely unknown due in part to current technical challenges in rapid isolation, molecular classification and comprehensive analysis of exosomes. Here we developed a new microfluidic approach to streamline and expedite the exosome analysis pipeline by integrating specific immunoisolation and targeted protein analysis of circulating exosomes. Compared to the conventional methods, our approach enables selective subpopulation isolation and quantitative detection of surface and intravesicular biomarkers directly from a minimally invasive amount of plasma samples (30 ?L) within ~100 min with markedly improved detection sensitivity. Using this device, we demonstrated phenotyping of exosome subpopulations by targeting a panel of common exosomal and tumor-specific markers and multiparameter analyses of intravesicular biomarkers in the selected subpopulation. We were able to assess the total expression and phosphorylation levels of IGF-1R in non-small-cell lung cancer patients by probing plasma exosomes as a non-invasive alternative to conventional tissue biopsy. We foresee that the microfluidic exosome analysis platform will form the basis for critically needed infrastructures for advancing the biology and clinical utilization of exosomes. (hide) EV-METRIC 44% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: CD63/ CD81/ CD9 non-EV: None Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ CD9 Fluorescent NTA Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV140026	1/1	Mus musculus	Cell culture supernatant	0.45 µm filter dUC Sucrose-DG UF	Han KY	2014	44%
Study summary Full title Evidence for the involvement of MMP14 in MMP2 processing and recruitment in exosomes of corneal fibroblasts All authors Han KY, Dugas-Ford J, Seiki M, Chang JH, Azar DT Journal Invest Ophthalmol Vis Sci Abstract PURPOSE: Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlyin (show more...)PURPOSE: Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlying mechanisms are poorly understood. In this study, we investigated exosomal transport of MMP14 and its target, MMP2, from corneal fibroblasts to vascular endothelial cells as a possible mechanism governing MMP14 activity in corneal angiogenesis. METHODS: We isolated MMP14-containing exosomes from corneal fibroblasts by sucrose density gradient and evaluated exosome content and purity by Western blot analysis. We then investigated exosome transport in vitro from corneal fibroblasts to two populations of vascular endothelial cells, human umbilical vein endothelial cells (HUVECs) and calf pulmonary artery endothelial cells (CPAECs). Western blot analysis and gelatin zymography were used to determine levels of MMP14 and MMP2, respectively, in exosomal fractions derived from cultured wild-type, MMP14 enzymatic domain-deficient (MMP14?exon4), and MMP14-null corneal fibroblasts. RESULTS: Matrix metalloproteinase 14-containing exosomes isolated from corneal fibroblasts were readily taken up in vitro by HUVECs and CPAECs. We found that MMP14 was enriched in exosomal fractions of cultured corneal fibroblasts. Moreover, loss of the MMP14 enzymatic domain resulted in accumulation of pro-MMP2 protein in exosomes, whereas MMP2 was nearly undetectable in exosomes of MMP14-null fibroblasts. CONCLUSIONS: Our results indicate that exosomes secreted by corneal fibroblasts can transport proteins, including MMP14, to vascular endothelial cells. In addition, recruitment of MMP2 into corneal fibroblast exosomes is an active process that depends, at least in part, on the presence of MMP14. The role of exosomal MMP14 transport in corneal angiogenesis has important implications for therapeutic applications targeting angiogenic processes in the cornea. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.45 µm filter + dUC + Sucrose-DG + UF Protein markers EV: TSG101/ Beta-actin non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Density gradient Density medium Sucrose Lowest density fraction 5 Highest density fraction 40 Orientation Bottom-up Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Detected EV-associated proteins TSG101/ Beta-actin Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins Beta-actin Fluorescent NTA Characterization: Particle analysis

	EV140024	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	de Carvalho JV	2014	44%
Study summary Full title Nef Neutralizes the Ability of Exosomes from CD4+ T Cells to Act as Decoys during HIV-1 Infection All authors de Carvalho JV, de Castro RO, da Silva EZ, Silveira PP, da Silva-Januário ME, Arruda E, Jamur MC, Oliver C, Aguiar RS, daSilva LL Journal PLoS One Abstract Nef is an HIV-1 accessory protein that promotes viral replication and pathogenesis. A key function o (show more...)Nef is an HIV-1 accessory protein that promotes viral replication and pathogenesis. A key function of Nef is to ensure sustained depletion of CD4 and MHC-I molecules in infected cells by inducing targeting of these proteins to multivesicular bodies (MVBs), and ultimately to lysosomes for degradation. Nef also affects cellular secretory routes promoting its own secretion via exosomes. To better understand the effects of Nef on the exocytic pathway, we investigated whether this viral factor modifies the composition of exosomes released by T lymphocytes. We showed that both CD4 and MHC-I molecules are secreted in exosomes from T cells and that the expression of Nef reduces the amount of these proteins in exosomes. To investigate the functional role for this novel activity of Nef, we performed in vitro HIV-1 infection assays in the presence of distinct populations of exosomes. We demonstrated that exosomes released by CD4+ T cells, but not CD4- T cells, efficiently inhibit HIV-1 infection in vitro. Because CD4 is the main receptor for HIV-1 infection, these results suggest that CD4 molecules displayed on the surface of exosom

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