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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140047	2/2	Homo sapiens	NAY	(d)(U)C Filtration	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD63/ CD133/ MFGE8/ CD81/ Flotilin1/ Annexin2/ CD13 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ Flotilin1/ MFGE8/ CD13/ CD133/ Annexin2 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MFGE8/ CD13/ CD133/ Annexin2 Characterization: Particle analysis NTA EM EM-type cryo EM Image type Close-up

	EV140111	1/2	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 295 (pelleting) Protein markers EV: Annexin1/ Tubulin/ TSG101/ Alix non-EV: Proteomics no Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 295.0 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ Annexin1/ Tubulin ELISA Antibody details provided? No Detected EV-associated proteins Annexin1/ Tubulin Characterization: Particle analysis NTA EM EM-type transmission EM/ immune EM EM protein Annexin1 ; 14-3-3beta Image type Close-up, Wide-field

	EV140110	2/2	Mus musculus	NAY	(d)(U)C 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Adj. k-factor 122.2 (pelleting) / 89.21 (washing) Protein markers EV: TSG101/ CD63/ HSP90/ Alix/ Tnfr1/ HSP70/ Beta-actin/ Ago1/ CD9/ Ago2 non-EV: 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 122.2 Wash: Rotor Type TLA55 Wash: adjusted k-factor 89.21 Density gradient Lowest density fraction 0.32 Highest density fraction 2 Orientation Top-down Rotor type TLA55 Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ CD9/ HSP90/ HSP70/ TSG101/ Ago1/ Ago2/ Tnfr1/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins Ago1/ Ago2/ Tnfr1/ Beta-actin Characterization: Particle analysis NTA

	EV140108	1/1	Acholeplasma laidlawii	Mycoplasma	(d)(U)C DG Filtration 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Mycoplasma Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration UF Adj. k-factor 95.8 (pelleting) Protein markers EV: HSP20 non-EV: Proteomics yes Show all info Study aim Omics Sample Species Acholeplasma laidlawii Sample Type Mycoplasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type MLA80 Pelleting: adjusted k-factor 95.8 Density gradient 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 Antibody details provided? No Detected EV-associated proteins HSP20 ELISA Antibody details provided? No Detected EV-associated proteins HSP20 Characterization: Particle analysis EM EM-type transmission EM/ immune EM/ scanning EM/ atomic force EM EM protein HSP20 Image type Close-up, Wide-field

	EV140238	3/3	Mus musculus	NAY	(d)(U)C 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ GM1/ Flotilin1/ Actin/ Beta-amyloid 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Lowest density fraction 0.25 Highest density fraction 2.2999999999999998 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ Flotilin1/ GM1/ Beta-amyloid/ Actin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins GM1/ Beta-amyloid/ Actin Characterization: Particle analysis None

	EV140044	2/2	Rattus norvegicus/rattus	NAY	(d)(U)C Filtration	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD81/ AChE/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ AChE ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

	EV140043	1/3	Homo sapiens	NAY	(d)(U)C Filtration	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ AChE/ CD63 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Wash: volume per pellet (ml) 10 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101/ AChE Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140041	1/1	Homo sapiens	NAY	(d)(U)C DG	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: TSG101/ CD63 non-EV: 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101 Characterization: Particle analysis DLS EM EM-type atomic force EM Image type Close-up, Wide-field

	EV140099	1/1	Homo sapiens	NAY	(d)(U)C 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Adj. k-factor 101.8 (pelleting) / 101.8 (washing) Protein markers EV: CD81/ Flotilin1 non-EV: Aconitase 2/ Cell organelle protein/ GAPDH/ Calreticulin/ VDAC1/ Beta-actin Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 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 Antibody details provided? No Detected EV-associated proteins CD81/ Flotilin1 Detected contaminants Cell organelle protein/ "VDAC1/ Calreticulin/ Aconitase 2/ GAPDH/ Beta-actin" Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140011	1/2	Homo sapiens	BALF	(d)(U)C Filtration	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% (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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type BALF Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type BALF Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140011	2/2	Homo sapiens	Blood plasma	(d)(U)C Filtration	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% (77th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140092	1/2	Homo sapiens	NAY	(d)(U)C DG	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ TSG101/ 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Density gradient Only used for validation of main results Yes 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 Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ Flotillin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Flotillin Characterization: Particle analysis EM EM-type immune EM EM protein PTEN;Ndfip1 Image type Close-up

	EV140092	2/2	Homo sapiens	NAY	(d)(U)C DG	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ TSG101/ 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Density gradient Only used for validation of main results Yes 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 Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ Flotillin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Flotillin Characterization: Particle analysis EM EM-type immune EM EM protein PTEN;Ndfip1 Image type Close-up

	EV140038	1/1	Homo sapiens	NAY	(d)(U)C 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Adj. k-factor 156.9 (pelleting) / 126 (washing) Protein markers EV: HSP90/ Flotilin1/ 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: Rotor Type 90Ti Wash: adjusted k-factor 126.0 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ HSP90/ MHC2 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MHC2 Characterization: Particle analysis DLS EM EM-type cryo EM Image type Wide-field

	EV140203	1/1	Mus musculus	NAY	(d)(U)C DG Filtration	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 255.8 (pelleting) Protein markers EV: ADAM10/ CD63 non-EV: 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Density gradient Only used for validation of main results Yes 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 Antibody details provided? No Detected EV-associated proteins CD63/ ADAM10 ELISA Antibody details provided? No Detected EV-associated proteins ADAM10 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140088	1/1	Homo sapiens	NAY	(d)(U)C Filtration 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 156.9 (pelleting) / 93.46 (washing) Protein markers EV: Tubulin/ Alix/ Beta-actin/ GAPDH/ Syntenin non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: volume per pellet (ml) 6 Wash: Rotor Type 100Ti Wash: adjusted k-factor 93.46 Filtration steps 0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ Syntenin/ Beta-actin/ GAPDH/ Tubulin ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin/ GAPDH/ Tubulin Characterization: Particle analysis None

	EV140119	2/3	Homo sapiens	NAY	(d)(U)C DG Filtration	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 55.47 (pelleting) Protein markers EV: AChE/ CD63/ HSP70 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW55 Pelleting: adjusted k-factor 55.47 Density gradient Only used for validation of main results Yes Lowest density fraction 6 Highest density fraction 24 Orientation Top-down Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ AChE ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140119	3/3	Homo sapiens	NAY	(d)(U)C DG Filtration	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 195.3 (pelleting) Protein markers EV: AChE/ CD63/ HSP70 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 195.3 Density gradient Only used for validation of main results Yes Lowest density fraction 6 Highest density fraction 24 Orientation Top-down Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ AChE ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140031	1/2	Homo sapiens	Urine	(d)(U)C 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% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes / extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 6 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ Galectin3 Detected contaminants Tamm-Horsfall glycoprotein ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ Galectin3 Detected contaminants Tamm-Horsfall glycoprotein Characterization: Particle analysis None

	EV140031	2/2	Homo sapiens	Urine	(d)(U)C Filtration 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% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes / extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 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 Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ Galectin3 Detected contaminants Tamm-Horsfall glycoprotein ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ Galectin3 Detected contaminants Tamm-Horsfall glycoprotein Characterization: Particle analysis EM EM-type transmission EM/ scanning EM Image type Wide-field Report size (nm) Not reported

	EV140083	1/1	Homo sapiens	NAY	(d)(U)C DG Filtration	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 213.3 (pelleting) Protein markers EV: Flotillin2 non-EV: 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 240 Pelleting: rotor type 32Ti Pelleting: adjusted k-factor 213.3 Density gradient Only used for validation of main results Yes 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 Antibody details provided? No Detected EV-associated proteins Flotillin2 ELISA Antibody details provided? No Detected EV-associated proteins Flotillin2 Characterization: Particle analysis EM EM-type immune EM EM protein Wnt3a Image type Close-up, Wide-field

	EV140179	1/1	Homo sapiens	NAY	(d)(U)C DG 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG UF Protein markers EV: Beta-actin/ MHC2/ FasL 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60-240 Density gradient Only used for validation of main results Yes Lowest density fraction 1.03g/l Highest density fraction 1.27g/l Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins MHC2/ FasL/ MHC2/ Beta-actin Detected contaminants FasL ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ FasL/ MHC2/ Beta-actin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis None

	EV140008	1/2	Homo sapiens	NAY	(d)(U)C Filtration	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed Yes Pelleting: time(min) 120 Wash: volume per pellet (ml) 30 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD9/ TSG101 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV140008	2/2	Homo sapiens	Serum	(d)(U)C Filtration 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% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: TSG101/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 960 Wash: volume per pellet (ml) 11 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD9/ TSG101 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV140007	1/1	Mus musculus	NAY	(d)(U)C DG	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: HSP90/ CD63/ 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Density gradient Only used for validation of main results Yes Lowest density fraction 5 Highest density fraction 40 Orientation Bottom-up Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP90/ Syntenin Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140030	2/2	Mus musculus	NAY	(d)(U)C 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Nano(-sized) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Microfluidics UF Protein markers EV: Nanog/ Actin/ ICAM1 non-EV: 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Other Name other separation method Microfluidics Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Actin/ Nanog/ ICAM1 ELISA Antibody details provided? No Detected EV-associated proteins Actin/ Nanog/ ICAM1 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140006	1/1	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD81/ GAPDH/ Alix/ Syntenin/ Beta-actin/ CD9 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ CD81/ CD9/ Syntenin/ Beta-actin/ GAPDH Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin/ GAPDH Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140029	1/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 475.5 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 475.5 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	2/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 234.2 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 234.2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	3/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 156.9 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	4/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 117.9 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 117.9 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	5/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 93.96 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 93.96 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	6/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 78.45 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	7/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 475.5 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 475.5 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	8/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 234.2 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 234.2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	9/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 156.9 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	10/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 117.9 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 117.9 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	11/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 93.96 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 93.96 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140029	12/12	Homo sapiens	NAY	(d)(U)C	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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 78.45 (pelleting) Protein markers EV: CD81/ TSG101/ Syntenin 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Syntenin/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA

	EV140027	1/1	Homo sapiens	NAY	(d)(U)C 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. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: CD63/ CD98/ EphA2/ Cofilin/ Caveolin1/ 4F2 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 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Caveolin1/ Cofilin/ EphA2/ 4F2/ CD98 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Caveolin1/ Cofilin/ EphA2/ 4F2/ CD98 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein 4F2 Image type Close-up, Wide-field Report size (nm) 86.9+-4.7

	EV140123	2/2	Homo sapiens	Blood plasma	(d)(U)C DG Filtration SEC	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% (77th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration SEC Protein markers EV: CD81 non-EV: Proteomics no EV density (g/ml) 1.16-1.19 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Density gradient 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 Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD81 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140117	2/8	Homo sapiens	Blood plasma	(d)(U)C	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% (77th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD81/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV140024	1/1	Homo sapiens	NAY	(d)(U)C Filtration	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 exosomes can bind to envelope proteins of HIV-1 hindering virus interaction with target cells and infection. Importantly, CD4-depleted exosomes released by CD4+ T cells expressing Nef have a reduced capacity to inhibit HIV-1 infection in vitro. These results provide evidence that Nef promotes HIV-1 infection by reducing the expression of CD4 in exosomes from infected cells, besides the original role of Nef in reducing the CD4 levels at the cell surface. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: Alix/ CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM/ scanning EM Image type Close-up, Wide-field Report size (nm) Not reported

	EV140068	1/3	Homo sapiens	NAY	(d)(U)C Filtration	Cvjetkovic A	2014	44%
Study summary Full title The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles All authors Cvjetkovic A, Lötvall J, Lässer C Journal J Extracell Vesicles Abstract BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consi (show more...)BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consist of vesicle species ranging in size from 30 nm to 5 µm in diameter. These vesicles are most commonly isolated by differential centrifugations, which pellets particles based on their differential movement through the liquid medium in which they are immersed. Multiple parameters, including the utilization of different rotor types, can influence the yield and purity of isolated vesicles; however, the understanding of how these factors affect is limited. MATERIALS AND METHODS: Here, we compare the influence of multiple centrifugation parameters, including the use of swinging bucket and fixed angle rotors, as well as different centrifugation times, for the isolation of the smallest EVs, exosomes. In particular, we determine the yields of exosomal RNA and protein, as well as the nature of the isolated vesicles and possible protein contamination with methods such as electron microscopy, western blot and flow cytometry. RESULTS: Our results show that application of a specific g-force or rotation speed by itself does not predict the ability of pelleting exosomes, and that prolonged centrifugation times can achieve greater yields of exosomal RNA and protein, whereas very long centrifugation times result in excessive protein concentrations in the exosome pellet. CONCLUSION: In conclusion, rotor type, g-force and centrifugation times significantly influence exosome yield during centrifugation-based isolation procedures, and current commonly recommended isolation protocols may not be fully optimized for yield and purity of exosomes. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 132.9 (pelleting) Protein markers EV: Calnexin/ CD81/ TSG101/ CD63/ CD9 non-EV: Cell organelle protein Proteomics no TEM measurements 53(median) Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 132.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ CD9/ TSG101/ Calnexin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes

	EV140068	3/3	Homo sapiens	NAY	(d)(U)C Filtration	Cvjetkovic A	2014	44%
Study summary Full title The influence of rotor type and centrifugation time on the yield and purity of extracellular vesicles All authors Cvjetkovic A, Lötvall J, Lässer C Journal J Extracell Vesicles Abstract BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consi (show more...)BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consist of vesicle species ranging in size from 30 nm to 5 µm in diameter. These vesicles are most commonly isolated by differential centrifugations, which pellets particles based on their differential movement through the liquid medium in which they are immersed. Multiple parameters, including the utilization of different rotor types, can influence the yield and purity of isolated vesicles; however, the understanding of how these factors affect is limited. MATERIALS AND METHODS: Here, we compare the influence of multiple centrifugation parameters, including the use of swinging bucket and fixed angle rotors, as well as different centrifugation times, for the isolation of the smallest EVs, exosomes. In particular, we determine the yields of exosomal RNA and protein, as well as the nature of the isolated vesicles and possible protein contamination with methods such as electron microscopy, western blot and flow cytometry. RESULTS: Our results show that application of a specific g-force or rotation speed by itself does not predict the ability of pelleting exosomes, and that prolonged centrifugation times can achieve greater yields of exosomal RNA and protein, whereas very long centrifugation times result in excessive protein concentrations in the exosome pellet. CONCLUSION: In conclusion, rotor type, g-force and centrifugation times significantly influence exosome yield during centrifugation-based isolation procedures, and current commonly recommended isolation protocols may not be fully optimized for yield and purity of exosomes. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 216.9 (pelleting) Protein markers EV: Calnexin/ CD81/ TSG101/ CD63/ CD9 non-EV: Cell organelle protein Proteomics no TEM measurements 53(median) Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 114 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 216.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ CD9/ TSG101/ Calnexin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Calnexin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes

	EV140067	1/1	Mus musculus Rattus norvegicus/rattus	NAY	(d)(U)C DG Filtration	Chivet M	2014	44%
Study summary Full title Exosomes secreted by cortical neurons upon glutamatergic synapse activation specifically interact with neurons All authors Chivet M, Javalet C, Laulagnier K, Blot B, Hemming FJ, Sadoul R Journal J Extracell Vesicles Abstract Exosomes are nano-sized vesicles of endocytic origin released into the extracellular space upon fusi (show more...)Exosomes are nano-sized vesicles of endocytic origin released into the extracellular space upon fusion of multivesicular bodies with the plasma membrane. Exosomes represent a novel mechanism of cell-cell communication allowing direct transfer of proteins, lipids and RNAs. In the nervous system, both glial and neuronal cells secrete exosomes in a way regulated by glutamate. It has been hypothesized that exosomes can be used for interneuronal communication implying that neuronal exosomes should bind to other neurons with some kind of specificity. Here, dissociated hippocampal cells were used to compare the specificity of binding of exosomes secreted by neuroblastoma cells to that of exosomes secreted by cortical neurons. We found that exosomes from neuroblastoma cells bind indiscriminately to neurons and glial cells and could be endocytosed preferentially by glial cells. In contrast, exosomes secreted from stimulated cortical neurons bound to and were endocytosed only by neurons. Thus, our results demonstrate for the first time that exosomes released upon synaptic activation do not bind to glial cells but selectively to other neurons suggesting that they can underlie a novel aspect of interneuronal communication. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 256 (pelleting) Protein markers EV: Alix/ Flotilin1/ CD63 non-EV: Proteomics no EV density (g/ml) 1.09-1.14 Show all info Study aim Function Sample Species Mus musculus / Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Density gradient Only used for validation of main results Yes Lowest density fraction 0.21099999999999999 Highest density fraction 1.4 Orientation Top-down Rotor type SW41 Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ Flotilin1 Characterization: Particle analysis EM EM-type immune EM EM protein CD63 Image type Close-up

	EV140147	1/3	Homo sapiens	Urine	(d)(U)C	Cheng L	2014	44%
Study summary Full title Characterization and deep sequencing analysis of exosomal and non-exosomal miRNA in human urine All authors Cheng L, Sun X, Scicluna BJ, Coleman BM, Hill AF Journal Kidney Int Abstract Micro RNAs (miRNAs) have been shown to circulate in biological fluids and are enclosed in vesicles s (show more...)Micro RNAs (miRNAs) have been shown to circulate in biological fluids and are enclosed in vesicles such as exosomes; they are present in urine and represent a noninvasive methodology to detect biomarkers for diagnostic testing. The low abundance of RNA in urine creates difficulties in its isolation, of which exosomal miRNA is a small fraction, making downstream RNA assays challenging. Here, we investigate methods to maximize exosomal isolation and RNA yield for next-generation deep sequencing. Upon characterizing exosomal proteins and total RNA content in urine, several commercially available kits were tested for their RNA extraction efficiency. We subsequently used the methods with the highest miRNA content to profile baseline miRNA expression using next-generation deep sequencing. Comparisons of miRNA profiles were also made with exosomes isolated by differential ultracentrifugation methodology and a commercially available column-based protocol. Overall, miRNAs were found to be significantly enriched and intact in urine-derived exosomes compared with cell-free urine. The presence of other noncoding RNAs such as small nuclear and small nucleolar RNA in the exosomes, in addition to coding sequences related to kidney and bladder conditions, was also detected. Our study extensively characterizes the RNA content of exosomes isolated from urine, providing the potential to identify miRNA biomarkers in human urine. (hide) EV-METRIC 44% (80th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 78.45 (pelleting) Protein markers EV: TSG101/ CD63/ Flotillin non-EV: Cell organelle protein Proteomics no Show all info Study aim Omics Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 65 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 78.45 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ Flotillin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Flotillin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV140020	1/1	Homo sapiens	NAY	(d)(U)C	Ahmed W	2014	44%
Study summary Full title Epstein-Barr virus-encoded small RNAs (EBERs) are present in fractions related to exosomes released by EBV-transformed cells All authors Ahmed W, Philip PS, Tariq S, Khan G Journal PLoS One Abstract Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a number of human malignancies (show more...)Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a number of human malignancies of epithelial and lymphoid origin. However, the mechanism of oncogenesis is unclear. A number of viral products, including EBV latent proteins and non-protein coding RNAs have been implicated. Recently it was reported that EBV-encoded small RNAs (EBERs) are released from EBV infected cells and they can induce biological changes in cells via signaling from toll-like receptor 3. Here, we investigated if these abundantly expressed non-protein coding EBV RNAs (EBER-1 and EBER-2) are excreted from infected cells in exosomal fractions. Using differential ultracentrifugation we isolated exosomes from three EBV positive cell lines (B95-8, EBV-LCL, BL30-B95-8), one EBER-1 transfected cell line (293T-pHEBo-E1) and two EBV-negative cell lines (BL30, 293T-pHEBo). The identity of purified exosomes was determined by electron microscopy and western blotting for CD63. The presence of EBERs in cells, culture supernatants and purified exosomal fractions was determined using RT-PCR and confirmed by sequencing. Purified exosomal fractions were also tested for the presence of the EBER-1-binding protein La, using western blotting. Both EBER-1 and EBER-2 were found to be present not only in the culture supernatants, but also in the purified exosome fractions of all EBV-infected cell lines. EBER-1 could also be detected in exosomal fractions from EBER-1 transfected 293T cells whilst the fractions from vector only transfectants were clearly negative. Furthermore, purified exosomal fractions also contained the EBER-binding protein (La), supporting the notion that EBERs are most probably released from EBV infected cells in the form of EBER-La complex in exosomes. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 256 (pelleting) Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140130	1/2	Homo sapiens	NAY	(d)(U)C DG	Fong MY	2014	43%
Study summary Full title Breast-cancer-secreted ?miR-122 reprograms ?glucose metabolism in premetastatic niche to promote metastasis All authors Fong MY, Zhou W, Liu L, Alontaga AY, Chandra M, Ashby J, Chow A, O'Connor ST, Li S, Chin AR, Somlo G, Palomares M, Li Z, Tremblay JR, Tsuyada A, Sun G, Reid MA, Wu X, Swiderski P, Ren X, Shi Y, Kong M, Zhong W, Chen Y, Wang SE Journal Nat Cell Biol Abstract Reprogrammed glucose metabolism as a result of increased glycolysis and glucose uptake is a hallmark (show more...)Reprogrammed glucose metabolism as a result of increased glycolysis and glucose uptake is a hallmark of cancer. Here we show that cancer cells can suppress glucose uptake by non-tumour cells in the premetastatic niche, by secreting vesicles that carry high levels of the miR-122 microRNA. High miR-122 levels in the circulation have been associated with metastasis in breast cancer patients, and we show that cancer-cell-secreted miR-122 facilitates metastasis by increasing nutrient availability in the premetastatic niche. Mechanistically, cancer-cell-derived miR-122 suppresses glucose uptake by niche cells in vitro and in vivo by downregulating the glycolytic enzyme pyruvate kinase. In vivo inhibition of miR-122 restores glucose uptake in distant organs, including brain and lungs, and decreases the incidence of metastasis. These results demonstrate that, by modifying glucose utilization by recipient premetastatic niche cells, cancer-derived extracellular miR-122 is able to reprogram systemic energy metabolism to facilitate disease progression. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: non-EV: Proteomics no TEM measurements 73.09 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Top-down Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field Report size (nm) 73.09

	EV140051	1/1	Cryptococcus neoformans	NAY	(d)(U)C Filtration UF	Wolf JM	2014	43%
Study summary Full title Interaction of Cryptococcus neoformans extracellular vesicles with the cell wall All authors Wolf JM, Espadas-Moreno J, Luque-Garcia JL, Casadevall A Journal Eukaryot Cell Abstract Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including vir (show more...)Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to trap vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition. (hide) EV-METRIC 43% (81st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: Urease/ Phosphatase/ Laccase non-EV: Proteomics yes Show all info Study aim Biogenesis/Sorting Sample Species Cryptococcus neoformans Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Urease/ Phosphatase/ Laccase ELISA Antibody details provided? No Detected EV-associated proteins Urease/ Phosphatase/ Laccase Characterization: Particle analysis DLS EM EM-type transmission EM/ cryo EM/ scanning EM Image type Close-up, Wide-field Report size (nm) Not reported

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