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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140286	1/4	Homo sapiens	NAY	(d)(U)C	Hutcheson JD	2014	43%
Study summary Full title Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol All authors Hutcheson JD, Goettsch C, Pham T, Iwashita M, Aikawa M, Singh SA, Aikawa E Journal J Extracell Vesicles Abstract Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have rece (show more...)Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have received increased attention for their role in mediating vascular calcification, a major predictor of cardiovascular morbidity and mortality. However, little is known about the difference between this pathologic vesicle population and other EVs that contribute to physiological cellular processes. One major challenge that hinders research into these differences is the inability to selectively isolate calcifying EVs from other vesicle populations. In this study, we hypothesized that the formation of mineral within calcifying EVs would increase the density of the vesicles such that they would pellet at a faster rate during ultracentrifugation. We show that after 10 min of ultracentrifugation at 100,000×g, calcifying EVs are depleted from the conditioned media of calcifying coronary artery smooth muscle cells and are enriched in the pelleted portion. We utilized mass spectrometry to establish functional proteomic differences between the calcifying EVs enriched in the 10 min ultracentrifugation compared to other vesicle populations preferentially pelleted by longer ultracentrifugation times. The procedures established in this study will allow us to enrich the vesicle population of interest and perform advanced proteomic analyses to find subtle differences between calcifying EVs and other vesicle populations that may be translated into therapeutic targets for vascular calcification. Finally, we will show that the differences in ultracentrifugation times required to pellet the vesicle populations can also be used to estimate physical differences between the vesicles. (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 Adj. k-factor 41.45 (pelleting) Protein markers EV: non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 10 Pelleting: rotor type TLA120.2 Pelleting: adjusted k-factor 41.45 Characterization: Protein analysis Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140286	2/4	Homo sapiens	NAY	(d)(U)C	Hutcheson JD	2014	43%
Study summary Full title Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol All authors Hutcheson JD, Goettsch C, Pham T, Iwashita M, Aikawa M, Singh SA, Aikawa E Journal J Extracell Vesicles Abstract Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have rece (show more...)Calcifying extracellular vesicles (EVs) released from cells within atherosclerotic plaques have received increased attention for their role in mediating vascular calcification, a major predictor of cardiovascular morbidity and mortality. However, little is known about the difference between this pathologic vesicle population and other EVs that contribute to physiological cellular processes. One major challenge that hinders research into these differences is the inability to selectively isolate calcifying EVs from other vesicle populations. In this study, we hypothesized that the formation of mineral within calcifying EVs would increase the density of the vesicles such that they would pellet at a faster rate during ultracentrifugation. We show that after 10 min of ultracentrifugation at 100,000×g, calcifying EVs are depleted from the conditioned media of calcifying coronary artery smooth muscle cells and are enriched in the pelleted portion. We utilized mass spectrometry to establish functional proteomic differences between the calcifying EVs enriched in the 10 min ultracentrifugation compared to other vesicle populations preferentially pelleted by longer ultracentrifugation times. The procedures established in this study will allow us to enrich the vesicle population of interest and perform advanced proteomic analyses to find subtle differences between calcifying EVs and other vesicle populations that may be translated into therapeutic targets for vascular calcification. Finally, we will show that the differences in ultracentrifugation times required to pellet the vesicle populations can also be used to estimate physical differences between the vesicles. (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 Adj. k-factor 41.45 (pelleting) Protein markers EV: non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 20 Pelleting: rotor type TLA120.2 Pelleting: adjusted k-factor 41.45 Characterization: Protein analysis Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140100	1/1	Homo sapiens	Milk	(d)(U)C DG Filtration	Torregrosa Paredes P	2014	43%
Study summary Full title Differences in exosome populations in human breast milk in relation to allergic sensitization and lifestyle All authors Torregrosa Paredes P, Gutzeit C, Johansson S, Admyre C, Stenius F, Alm J, Scheynius A, Gabrielsson S Journal Allergy Abstract BACKGROUND: Breast-feeding has many beneficial effects on the developing immune system of the newbor (show more...)BACKGROUND: Breast-feeding has many beneficial effects on the developing immune system of the newborn. Breast milk contains immunoregulatory factors, such as nano-sized vesicles named exosomes. This study aimed at characterizing breast milk exosomes from human early milk and mature milk and to investigate whether allergic sensitization and an anthroposophic lifestyle could influence the exosome profile. METHODS: Breast milk was collected from 22 mothers at day 3-8 and from 61 mothers at 2 months postpartum, all part of the ALADDIN birth cohort. Isolated exosomes were captured on anti-MHC-class II- or anti-CD63 beads and analyzed by flow cytometry. Exosomal phenotype was related to lifestyle and allergic sensitization of the mothers, and sensitization of the child at 2 years of age. RESULTS: We found a higher content of exosomes in early milk compared with mature milk. Early milk exosomes were enriched in HLA-DR molecules and displayed significantly lower levels of HLA-ABC compared with those in mature milk. Phenotypically different subpopulations of exosomes were found in mature milk. Significantly lower levels of MUC1 were detected on CD63-enriched exosomes from sensitized mothers compared with nonsensitized. Furthermore, women with an anthroposophic lifestyle had significantly lower MUC1 expression on their HLA-DR-enriched milk exosomes and up-regulated levels of CD63 on CD63-enriched exosomes compared with nonanthroposophic mothers. Notably, mothers whose children developed sensitization had an increased amount of HLA-ABC on their milk exosomes enriched for CD63. CONCLUSIONS: The phenotype of exosomes in breast milk varies with maternal sensitization and lifestyle, which might influence allergy development in the child. (hide) EV-METRIC 43% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: CD81/ CD63/ MUC1/ MHC2/ CD36 non-EV: Proteomics no EV density (g/ml) 1.12-1.18 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Milk 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 0.25 Highest density fraction 2 Orientation Top-down Speed (g) 200000 Filtration steps > 0.45 µm, 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Western Blot Antibody details provided? No Detected EV-associated proteins MHC2/ CD36/ MUC1/ MHC2 ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ CD36/ MUC1/ MHC2 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis NTA

	EV140080	1/2	Homo sapiens	Urine	(d)(U)C DG	Hogan MC	2014	43%
Study summary Full title Identification of Biomarkers for PKD1 Using Urinary Exosomes All authors Hogan MC, Bakeberg JL, Gainullin VG, Irazabal MV, Harmon AJ, Lieske JC, Charlesworth MC, Johnson KL, Madden BJ, Zenka RM, McCormick DJ, Sundsbak JL, Heyer CM, Torres VE, Harris PC, Ward CJ Journal J Am Soc Nephrol Abstract Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals (show more...)Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of ESRD. Affected individuals inherit a defective copy of either PKD1 or PKD2, which encode polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 and PC2 are secreted on urinary exosome-like vesicles (ELVs) (100-nm diameter vesicles), in which PC1 is present in a cleaved form and may be complexed with PC2. Here, label-free quantitative proteomic studies of urine ELVs in an initial discovery cohort (13 individuals with PKD1 mutations and 18 normal controls) revealed that of 2008 ELV proteins, 9 (0.32%) were expressed at significantly different levels in samples from individuals with PKD1 mutations compared to controls (P<0.03). In samples from individuals with PKD1 mutations, levels of PC1 and PC2 were reduced to 54% (P<0.02) and 53% (P<0.001), respectively. Transmembrane protein 2 (TMEM2), a protein with homology to fibrocystin, was 2.1-fold higher in individuals with PKD1 mutations (P<0.03). The PC1/TMEM2 ratio correlated inversely with height-adjusted total kidney volume in the discovery cohort, and the ratio of PC1/TMEM2 or PC2/TMEM2 could be used to distinguish individuals with PKD1 mutations from controls in a confirmation cohort. In summary, results of this study suggest that a test measuring the urine exosomal PC1/TMEM2 or PC2/TMEM2 ratio may have utility in diagnosis and monitoring of polycystic kidney disease. Future studies will focus on increasing sample size and confirming these studies. The data were deposited in the ProteomeXchange (identifier PXD001075). (hide) EV-METRIC 43% (76th 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 DG Adj. k-factor 168.7 (pelleting) Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.046-1.064 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine 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) 120 Pelleting: rotor type T647.5 Pelleting: adjusted k-factor 168.7 Density gradient Lowest density fraction 5 Highest density fraction 30 Orientation Top-down Rotor type Surespin Speed (g) 200000 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140266	3/6	Homo sapiens	Blood plasma	(d)(U)C	Chevillet JR	2014	43%
Study summary Full title Quantitative and stoichiometric analysis of the microRNA content of exosomes All authors Chevillet JR, Kang Q, Ruf IK, Briggs HA, Vojtech LN, Hughes SM, Cheng HH, Arroyo JD, Meredith EK, Gallichotte EN, Pogosova-Agadjanyan EL, Morrissey C, Stirewalt DL, Hladik F, Yu EY, Higano CS, Tewari M Journal Proc Natl Acad Sci U S A Abstract Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and (show more...)Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes. (hide) EV-METRIC 43% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type 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 Adj. k-factor 115.5 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting 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) 70 Pelleting: rotor type SW55 Pelleting: adjusted k-factor 115.5 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up, Wide-field

	EV140135	1/1	Dicrocoelium dendriticum	Parasite	(d)(U)C Filtration	Bernal D	2014	43%
Study summary Full title Surface analysis of Dicrocoelium dendriticum. The molecular characterization of exosomes reveals the presence of miRNAs All authors Bernal D, Trelis M, Montaner S, Cantalapiedra F, Galiano A, Hackenberg M, Marcilla A Journal J Proteomics Abstract With the aim of characterizing the molecules involved in the interaction of Dicrocoelium dendriticum (show more...)With the aim of characterizing the molecules involved in the interaction of Dicrocoelium dendriticum adults and the host, we have performed proteomic analyses of the external surface of the parasite using the currently available datasets including the transcriptome of the related species Echinostoma caproni. We have identified 182 parasite proteins on the outermost surface of D. dendriticum. The presence of exosome-like vesicles in the ESP of D. dendriticum and their components has also been characterized. Using proteomic approaches, we have characterized 84 proteins in these vesicles. Interestingly, we have detected miRNA in D. dendriticum exosomes, thus representing the first report of miRNA in helminth exosomes.BIOLOGICAL SIGNIFICANCE: In order to identify potential targets for intervention against parasitic helminths, we have analyzed the surface of the parasitic helminth Dicrocoelium dendriticum. Along with the proteomic analyses of the outermost layer of the parasite, our work describes the molecular characterization of the exosomes of D. dendriticum. Our proteomic data confirm the improvement of protein identification from non-model organisms like helminths, when using different search engines against a combination of available databases. In addition, this work represents the first report of miRNAs in parasitic helminth exosomes. These vesicles can pack specific proteins and RNAs providing stability and resistance to RNAse digestion in body fluids, and provide a way to regulate host-parasite interplay. The present data should provide a solid foundation for the development of novel methods to control this non-model organism and related parasites. This article is part of a Special Issue entitled: Proteomics of non-model organisms. (hide) EV-METRIC 43% (60th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Parasite Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 74.34 (pelleting) / 74.34 (washing) Protein markers EV: non-EV: Proteomics yes Show all info Study aim Omics Sample Species Dicrocoelium dendriticum Sample Type Parasite Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type TLA55 Pelleting: adjusted k-factor 74.34 Wash: Rotor Type TLA55 Wash: adjusted k-factor 74.34 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140120	5/7	Homo sapiens	NAY	(d)(U)C Filtration Vn peptides	Ghosh A	2014	38%
Study summary Full title Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins All authors Ghosh A, Davey M, Chute IC, Griffiths SG, Lewis S, Chacko S, Barnett D, Crapoulet N, Fournier S, Joy A, Caissie MC, Ferguson AD, Daigle M, Meli MV, Lewis SM, Ouellette RJ Journal PLoS One Abstract Recent studies indicate that extracellular vesicles are an important source material for many clinic (show more...)Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 Vn peptides Protein markers EV: HSP70/ HSP90/ CD63/ GAPDH non-EV: Proteomics yes Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Other Name other separation method Vn peptides Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP90/ HSP70/ GAPDH ELISA Antibody details provided? No Detected EV-associated proteins GAPDH Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140104	2/3	Homo sapiens	Milk	(d)(U)C DG	Zonneveld MI	2014	38%
Study summary Full title Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures All authors Zonneveld MI, Brisson AR, van Herwijnen MJ, Tan S, van de Lest CH, Redegeld FA, Garssen J, Wauben MH, Nolte-'t Hoen EN Journal J Extracell Vesicles Abstract Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an importan (show more...)Extracellular vesicles (EV) in breast milk carry immune relevant proteins and could play an important role in the instruction of the neonatal immune system. To further analyze these EV and to elucidate their function it is important that native populations of EV can be recovered from (stored) breast milk samples in a reproducible fashion. However, the impact of isolation and storage procedures on recovery of breast milk EV has remained underexposed. Here, we aimed to define parameters important for EV recovery from fresh and stored breast milk. To compare various protocols across different donors, breast milk was spiked with a well-defined murine EV population. We found that centrifugation of EV down into density gradients largely improved density-based separation and isolation of EV, compared to floatation up into gradients after high-force pelleting of EV. Using cryo-electron microscopy, we identified different subpopulations of human breast milk EV and a not previously described population of lipid tubules. Additionally, the impact of cold storage on breast milk EV was investigated. We determined that storing unprocessed breast milk at -80°C or 4°C caused death of cells present in breast milk, leading to contamination of the breast milk EV population with storage-induced EV. Here, an alternative method is proposed to store breast milk samples for EV analysis at later time points. The proposed adaptations to the breast milk storage and EV isolation procedures can be applied for EV-based biomarker profiling of breast milk and functional analysis of the role of breast milk EV in the development of the neonatal immune system. (hide) EV-METRIC 38% (62nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin NAY Focus vesicles 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 Protein markers EV: CD63/ CD9 non-EV: Proteomics no EV density (g/ml) 1.12-1.28 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Lowest density fraction 0.4 Highest density fraction 2.5 Orientation Bottom-up Rotor type SW60 Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD9 Characterization: Particle analysis None

	EV140103	1/1	Homo sapiens	NAY	(d)(U)C DG UF	Yoon YJ	2014	38%
Study summary Full title Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways All authors Yoon YJ, Kim DK, Yoon CM, Park J, Kim YK, Roh TY, Gho YS Journal PLoS One Abstract Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as ex (show more...)Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1) activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference-mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 UF Protein markers EV: CD81/ CD63 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.098 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Density gradient Lowest density fraction 5 Highest density fraction 30 Orientation Bottom-up Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Detected contaminants Cell organelle protein Characterization: Particle analysis None

	EV140045	2/2	Homo sapiens	NAY	(d)(U)C DG	Webber J	2014	38%
Study summary Full title Proteomics analysis of cancer exosomes using a novel modified aptamer-based array (SOMAscan) platform All authors Webber J, Stone TC, Katilius E, Smith BC, Gordon B, Mason MD, Tabi Z, Brewis IA, Clayton A Journal Mol Cell Proteomics Abstract We have used a novel affinity-based proteomics technology to examine the protein signature of small (show more...)We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes. The technology uses a new class of protein binding reagents called SOMAmers® (slow off-rate modified aptamers) and allows the simultaneous precise measurement of over 1000 proteins. Exosomes were highly purified from the Du145 prostate cancer cell line, by pooling selected fractions from a continuous sucrose gradient (within the density range of 1.1 to 1.2 g/ml), and examined under standard conditions or with additional detergent treatment by the SOMAscan™ array (version 3.0). Lysates of Du145 cells were also prepared, and the profiles were compared. Housekeeping proteins such as cyclophilin-A, LDH, and Hsp70 were present in exosomes, and we identified almost 100 proteins that were enriched in exosomes relative to cells. These included proteins of known association with cancer exosomes such as MFG-E8, integrins, and MET, and also those less widely reported as exosomally associated, such as ROR1 and ITIH4. Several proteins with no previously known exosomal association were confirmed as exosomally expressed in experiments using individual SOMAmer® reagents or antibodies in micro-plate assays. Western blotting confirmed the SOMAscan™-identified enrichment of exosomal NOTCH-3, L1CAM, RAC1, and ADAM9. In conclusion, we describe here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmer®-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: CD81/ PSMA/ CD63/ CD9/ MHC1 non-EV: Proteomics yes EV density (g/ml) 1.12-1.17 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Lowest density fraction 0.2 Highest density fraction 2.5 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins PSMA/ MHC1 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ PSMA/ MHC1 Characterization: Particle analysis NTA

	EV140040	1/1	Homo sapiens	Endometriomas and simple cysts fluid	(d)(U)C ExoQuick	Texidó L	2014	38%
Study summary Full title Ecto-nucleotidases activities in the contents of ovarian endometriomas: potential biomarkers of endometriosis All authors Texidó L, Romero C, Vidal A, García-Valero J, Fernández Montoli ME, Baixeras N, Condom E, Ponce J, García-Tejedor A, Martín-Satué M Journal Mediators Inflamm Abstract Endometriosis, defined as the growth of endometrial tissue outside the uterus, is a common gynecolog (show more...)Endometriosis, defined as the growth of endometrial tissue outside the uterus, is a common gynecologic condition affecting millions of women worldwide. It is an inflammatory, estrogen-dependent complex disorder, with broad symptomatic variability, pelvic pain, and infertility being the main characteristics. Ovarian endometriomas are frequently developed in women with endometriosis. Late diagnosis is one of the main problems of endometriosis; thus, it is important to identify biomarkers for early diagnosis. The aim of the present work is to evaluate the ecto-nucleotidases activities in the contents of endometriomas. These enzymes, through the regulation of extracellular ATP and adenosine levels, are key enzymes in inflammatory processes, and their expression has been previously characterized in human endometrium. To achieve our objective, the echo-guided aspirated fluids of endometriomas were analyzed by evaluating the ecto-nucleotidases activities and compared with simple cysts. Our results show that enzyme activities are quantifiable in the ovarian cysts aspirates and that endometriomas show significantly higher ecto-nucleotidases activities than simple cysts (5.5-fold increase for ATPase and 20-fold for ADPase), thus being possible candidates for new endometriosis biomarkers. Moreover, we demonstrate the presence of ecto-nucleotidases bearing exosomes in these fluids. These results add up to the knowledge of the physiopathologic mechanisms underlying endometriosis and, open up a promising new field of study. (hide) EV-METRIC 38% (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 Endometriomas and simple cysts fluid 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 ExoQuick Protein markers EV: CD39/ CD63/ CD73/ CD81/ HSP70/ CD9 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Endometriomas and simple cysts fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ CD9/ HSP70/ CD39/ CD73 ELISA Antibody details provided? No Detected EV-associated proteins CD39/ CD73 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140125	1/2	Homo sapiens	Blood plasma	(d)(U)C IAF	Shi M	2014	38%
Study summary Full title Plasma exosomal alpha-synuclein is likely CNS-derived and increased in Parkinson's disease All authors Shi M, Liu C, Cook TJ, Bullock KM, Zhao Y, Ginghina C, Li Y, Aro P, Dator R, He C, Hipp MJ, Zabetian CP, Peskind ER, Hu SC, Quinn JF, Galasko DR, Banks WA, Zhang J Journal Acta Neuropathol Abstract Extracellular ?-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a (show more...)Extracellular ?-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum ?-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived ?-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled ?-synuclein into mouse brain, that CSF ?-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of ?-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF ?-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal ?-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF ?-synuclein, a significant correlation between plasma exosomal ?-synuclein and disease severity (r = 0.176, p = 0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal ?-synuclein were comparable to those determined by CSF ?-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS ?-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically. (hide) EV-METRIC 38% (72nd 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 IAF Protein markers EV: Alix/ Alpha-synuclein/ L1CAM non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Immunoaffinity capture Selected surface protein(s) L1CAM, CD63 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ L1CAM/ Alpha-synuclein ELISA Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Alix/ L1CAM/ Alpha-synuclein Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein L1CAM Image type Close-up, Wide-field

	EV140037	1/1	Homo sapiens	NAY	(d)(U)C IAF Total Exosome Isolation	Oksvold MP	2014	38%
Study summary Full title Expression of B-cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells All authors Oksvold MP, Kullmann A, Forfang L, Kierulf B, Li M, Brech A, Vlassov AV, Smeland EB, Neurauter A, Pedersen KW Journal Clin Ther Abstract PURPOSE: Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found (show more...)PURPOSE: Exosomes are small (30- to 100-nm) vesicles secreted by all cell types in culture and found in most body fluids. A mean of 1 mL of blood serum, derived from healthy donors, contains approximately 10(12) exosomes. Depending on the disease, the number of exosomes can fluctuate. Concentration of exosomes in the bloodstream and all other body fluids is extremely high. Several B-cell surface antigens (CD19, CD20, CD22, CD23, CD24, CD37, CD40, and HLA-DR) and the common leukocyte antigen CD45 are interesting in terms of immunotherapy of hematologic malignant neoplasms. The established standard for exosome isolation is ultracentrifugation. However, this method cannot discriminate between exosome subpopulations and other nanovesicles. The main purpose of this study was to characterize CD81(+) and CD63(+) subpopulations of exosomes in terms of these surface markers after release from various types of B-cell lymphoma cell lines using an easy and reliable method of immunomagnetic separation. METHODS: Western blotting, flow cytometry, and electron microscopy were used to compare the total preenriched extracellular vesicle (EV) pool to each fraction of vesicles after specific isolation, using magnetic beads conjugated with antibodies raised against the exosome markers CD63 and CD81. FINDINGS: Magnetic bead-based isolation is a convenient method to study and compare subpopulations of exosomes released from B-cell lymphoma cells. The data indicated that the specifically isolated vesicles differed from the total preenriched EV pool. CD19, CD20, CD24, CD37, and HLA-DR, but not CD22, CD23, CD40, and CD45, are expressed on exosomes from B-cell lymphoma cell lines with large heterogeneity among the different B-cell lymphoma cell lines. Interestingly, these B-cell lymphoma-derived EVs are able to rescue lymphoma cells from rituximab-induced complement-dependent cytotoxicity. IMPLICATIONS: Distribution of exosomes that contain CD19, CD20, CD24, CD37, and HLA-DR may intercept immunotherapy directed against these antigens, which is important to be aware of for optimal treatment. The use of an immunomagnetic separation platform enables easy isolation and characterization of exosome subpopulations for further studies of the exosome biology to understand the potential for therapeutic and diagnostic use. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C IAF Total Exosome Isolation Protein markers EV: CD81/ CD63/ MHC2/ CD9 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation Immunoaffinity capture Selected surface protein(s) CD81, CD63 Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ MHC2 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MHC2 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 Wide-field

	EV140034	1/1	Homo sapiens	Serum	ExoQuick Filtration	Manterola L	2014	38%
Study summary Full title A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool All authors Manterola L, Guruceaga E, Gállego Pérez-Larraya J, González-Huarriz M, Jauregui P, Tejada S, Diez-Valle R, Segura V, Samprón N, Barrena C, Ruiz I, Agirre A, Ayuso A, Rodríguez J, González A, Xipell E, Matheu A, López de Munain A, Tuñón T, Zazpe I, García-Foncillas J, Paris S, Delattre JY, Alonso MM Journal Neuro Oncol Abstract BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and (show more...)BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. METHODS: To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. RESULTS: We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. CONCLUSIONS: Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker. (hide) EV-METRIC 38% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 ExoQuick Filtration Protein markers EV: Alix/ TSG101/ CD9/ LAMP1 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD9/ TSG101/ LAMP1 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins LAMP1 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV140193	2/3	Homo sapiens	Semen	(d)(U)C ExoQuick IAF	Madison MN	2014	38%
Study summary Full title Human semen contains exosomes with potent anti-HIV-1 activity All authors Madison MN, Roller RJ, Okeoma CM Journal Retrovirology Abstract BACKGROUND: Exosomes are membranous nanovesicles secreted into the extracellular milieu by diverse c (show more...)BACKGROUND: Exosomes are membranous nanovesicles secreted into the extracellular milieu by diverse cell types. Exosomes facilitate intercellular communication, modulate cellular pheno/genotype, and regulate microbial pathogenesis. Although human semen contains exosomes, their role in regulating infection with viruses that are sexually transmitted remains unknown. In this study, we used semen exosomes purified from healthy human donors to evaluate the role of exosomes on the infectivity of different strains of HIV-1 in a variety of cell lines. RESULTS: We show that human semen contains a heterologous population of exosomes, enriched in mRNA encoding tetraspanin exosomal markers and various antiviral factors. Semen exosomes are internalized by recipient cells and upon internalization, inhibit replication of a broad array of HIV-1 strains. Remarkably, the anti-HIV-1 activity of semen exosomes is specific to retroviruses because semen exosomes blocked replication of the murine AIDS (mAIDS) virus complex (LP-BM5). However, exosomes from blood had no effect on HIV-1 or LP-BM5 replication. Additionally, semen and blood exosomes had no effect on replication of herpes simplex virus; types 1 and 2 (HSV1 and HSV2). Mechanistic studies indicate that semen exosomes exert a post-entry block on HIV-1 replication by orchestrating deleterious effects on particle-associated reverse transcriptase activity and infectivity. CONCLUSIONS: These illuminating findings i) improve our knowledge of the cargo of semen exosomes, ii) reveal that semen exosomes possess anti-retroviral activity, and iii) suggest that semen exosome-mediated inhibition of HIV-1 replication may provide novel opportunities for the development of new therapeutics for HIV-1. (hide) EV-METRIC 38% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Semen 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 ExoQuick IAF Protein markers EV: CD81/ CD63/ GAPDH non-EV: Cell organelle protein Proteomics no TEM measurements 50-100 Show all info Study aim Function Sample Species Homo sapiens Sample Type Semen Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Immunoaffinity capture Selected surface protein(s) MHC2 Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ GAPDH Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins GAPDH Characterization: Particle analysis EM EM-type immune EM EM protein CD63 Image type Close-up Report size (nm) 50-100

	EV140076	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Goldie BJ	2014	38%
Study summary Full title Activity-associated miRNA are packaged in Map1b-enriched exosomes released from depolarized neurons All authors Goldie BJ, Dun MD, Lin M, Smith ND, Verrills NM, Dayas CV, Cairns MJ Journal Nucleic Acids Res Abstract Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity requi (show more...)Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity requiring complex mechanisms of post-transcriptional mRNA trafficking and regulation. Small non-coding miRNA are uniquely poised to support these functions by providing a nucleic-acid-based specificity component for universal-sequence-dependent RNA binding complexes. We investigated the subcellular distribution of these molecules in resting and potassium chloride depolarized human neuroblasts, and found both selective enrichment and depletion in neurites. Depolarization was associated with a neurite-restricted decrease in miRNA expression; a subset of these molecules was recovered from the depolarization medium in nuclease resistant extracellular exosomes. These vesicles were enriched with primate specific miRNA and the synaptic-plasticity-associated protein MAP1b. These findings further support a role for miRNA as neural plasticity regulators, as they are compartmentalized in neurons and undergo activity-associated redistribution or release into the extracellular matrix. (hide) EV-METRIC 38% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: Flotilin1/ LAMP1 non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method Filtration steps 0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ LAMP1 ELISA Antibody details provided? No Detected EV-associated proteins LAMP1 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140122	1/2	Homo sapiens	Urine	Filtration IAF filter	Echevarria J	2014	38%
Study summary Full title Microarray-based identification of lectins for the purification of human urinary extracellular vesicles directly from urine samples All authors Echevarria J, Royo F, Pazos R, Salazar L, Falcon-Perez JM, Reichardt NC Journal Chembiochem Abstract As cellular-derived vesicles largely maintain the biomolecule composition of their original tissue, (show more...)As cellular-derived vesicles largely maintain the biomolecule composition of their original tissue, exosomes, which are found in nearly all body fluids, have enormous potential as clinical disease markers. A major bottleneck in the development of exosome-based diagnostic assays is the challenging purification of these vesicles; this requires time-consuming and instrument-based procedures. We employed lectin arrays to identify potential lectins as probes for affinity-based isolation of exosomes from the urinary matrix. We found three lectins that showed specific interactions to vesicles and no (or only residual) interaction with matrix proteins. Based on these findings a bead-based method for lectin-based isolation of exosomes from urine was developed as a sample preparation step for exosome-based biomarker research. (hide) EV-METRIC 38% (73rd 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 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 Filtration IAF filter Protein markers EV: CD63/ Flotilin1/ AQP2/ CD13/ AQP1 non-EV: Tamm-Horsfall glycoprotein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Separation Method Immunoaffinity capture Selected surface protein(s) lectin Other Name other separation method filter Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Flotilin1/ AQP1/ AQP2/ CD13 Detected contaminants Tamm-Horsfall glycoprotein ELISA Antibody details provided? No Detected EV-associated proteins AQP1/ AQP2/ CD13 Characterization: Particle analysis EM EM-type cryo EM Image type Wide-field

	EV140023	1/1	Ovis aries	Serum	(d)(U)C ExoQuick	Cleys ER	2014	38%
Study summary Full title Identification of microRNAs in exosomes isolated from serum and umbilical cord blood, as well as placentomes of gestational day 90 pregnant sheep All authors Cleys ER, Halleran JL, McWhorter E, Hergenreder J, Enriquez VA, da Silveira JC, Bruemmer JE, Winger QA, Bouma GJ Journal Mol Reprod Dev Abstract Despite reports that circulating levels of maternal serum exosomes increase during pregnancy and tha (show more...)Despite reports that circulating levels of maternal serum exosomes increase during pregnancy and that placenta-specific microRNAs (miRNAs) have been identified in humans, little is known about exosomes and miRNAs during pregnancy in agriculture animals. In this study, we characterized the expression of 94 miRNAs in ovine placentomes at gestation day (GD) 90 by real-time PCR, and then investigated the presence of these miRNAs in exosome samples isolated from maternal jugular blood in non-pregnant ewes and at GD30 and GD90 and in umbilical blood collected at GD90. In maternal jugular exosome samples, 13 miRNAs were present in lower and 12 miRNAs were present in higher amounts at GD90 compared to non-pregnant (GD0) or GD30. Additionally, 12 miRNAs were present in higher amounts in umbilical venous exosomes compared to umbilical arterial exosomes; only miR-132 was lower in exosomes isolated from umbilical venous blood than from umbilical arterial blood. In placentome samples, miR-34c and miR135a abundance was higher in cotyledon tissue than in caruncle, while miR-183 and miR-379 amounts were higher in caruncle than cotyledon tissue. Only miR-379 was differentially expressed in all serum exosomes and placentome samples. Pathway analysis predicted that differentially expressed maternal serum exosomal miRNAs target Cellular Growth and Proliferation and Organ Development pathways, while umbilical serum exosomal and placentomes miRNAs were predicted to target cellular development and organismal/embryonic development. (hide) EV-METRIC 38% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 ExoQuick Protein markers EV: HSP70 non-EV: Cell organelle protein Proteomics no Show all info Study aim Omics Sample Species Ovis aries Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins HSP70 Detected contaminants Cell organelle protein Characterization: Particle analysis None

	EV140265	1/1	Mesocricetus auratus	NAY	(d)(U)C DC	Zeev-Ben-Mordehai T	2014	33%
Study summary Full title Extracellular Vesicles: A Platform for the Structure Determination of Membrane Proteins by Cryo-EM All authors Zeev-Ben-Mordehai T, Vasishtan D, Siebert CA, Whittle C, Grünewald K Journal Structure Abstract Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact mem (show more...)Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact membrane proteins natively anchored with the correct topology in genuine biological membranes. This approach circumvents the need to conduct tedious detergent screens for solubilization, purification, and reconstitution required in classical membrane protein studies. We have applied this method to three integral type I membrane proteins, namely the Caenorhabditis elegans cell-cell fusion proteins AFF-1 and EFF-1 and the glycoprotein B (gB) from Herpes simplex virus type 1 (HSV1). Electron cryotomography followed by subvolume averaging allowed the 3D reconstruction of EFF-1 and HSV1 gB in the membrane as well as an analysis of the spatial distribution and interprotein interactions on the membrane. MPEEVs have many applications beyond structural/functional investigations, such as facilitating the raising of antibodies, for protein-protein interaction assays or for diagnostics use, as biomarkers, and possibly therapeutics. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 DC Protein markers EV: non-EV: Proteomics yes Show all info Study aim Technical Sample Species Mesocricetus auratus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Characterization: Protein analysis Characterization: Particle analysis EM EM-type cryo EM Image type Close-up, Wide-field

	EV140116	1/1	Candida albicans	Yeast	(d)(U)C Filtration UF	Vargas G	2014	33%
Study summary Full title Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans All authors Vargas G, Rocha JD, Oliveira DL, Albuquerque PC, Frases S, Santos SS, Nosanchuk JD, Gomes AM, Medeiros LC, Miranda K, Sobreira TJ, Nakayasu ES, Arigi EA, Casadevall A, Guimaraes AJ, Rodrigues ML, Freire-de-Lima CG, Almeida IC, Nimrichter L Journal Cell Microbiol Abstract The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transpor (show more...)The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-?) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis. (hide) EV-METRIC 33% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Yeast Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: C. albicans antigens non-EV: Proteomics yes Show all info Study aim Omics Sample Species Candida albicans Sample Type Yeast Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps > 0.45 µm, Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins "C. albicans antigens" ELISA Antibody details provided? No Detected EV-associated proteins "C. albicans antigens" Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140290	1/3	Homo sapiens	NAY	(d)(U)C UF	van der Mijn JC	2014	33%
Study summary Full title Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer All authors van der Mijn JC, Sol N, Mellema W, Jimenez CR, Piersma SR, Dekker H, Schutte LM, Smit EF, Broxterman HJ, Skog J, Tannous BA, Wurdinger T, Verheul HM Journal J Extracell Vesicles Abstract BACKGROUND: Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in (show more...)BACKGROUND: Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors. METHODS: EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used. RESULTS: In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p<0.005). CONCLUSION: Phosphorylation of protein kinases in EVs reflects their phosphorylation in tumour cells. Total AKT protein levels may allow monitoring of kinase inhibitor responses in patients with cancer. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 256 (pelleting) Protein markers EV: Alix/ CD63 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum freeEV 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 SW32 Pelleting: adjusted k-factor 256.0 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63 Detected contaminants Cell organelle protein Characterization: Particle analysis None

	EV140258	2/3	Mus musculus	NAY	(d)(U)C DG Filtration	Ridder K	2014	33%
Study summary Full title Extracellular vesicle-mediated transfer of genetic information between the hematopoietic system and the brain in response to inflammation All authors Ridder K, Keller S, Dams M, Rupp AK, Schlaudraff J, Del Turco D, Starmann J, Macas J, Karpova D, Devraj K, Depboylu C, Landfried B, Arnold B, Plate KH, Höglinger G, Sültmann H, Altevogt P, Momma S Journal PLoS Biol Abstract Mechanisms behind how the immune system signals to the brain in response to systemic inflammation ar (show more...)Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 Adj. k-factor 97.39 (pelleting) Protein markers EV: ADAM10/ CD9 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 OR 1080 Pelleting: rotor type SW40 Pelleting: adjusted k-factor 97.39 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Rotor type SW40 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ ADAM10 ELISA Antibody details provided? No Detected EV-associated proteins ADAM10 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140255	1/1	Homo sapiens	NAY	(d)(U)C	Morhayim J	2014	33%
Study summary Full title Proteomic signatures of extracellular vesicles secreted by nonmineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth All authors Morhayim J, van de Peppel J, Demmers JA, Kocer G, Nigg AL, van Driel M, Chiba H, van Leeuwen JP Journal FASEB J Abstract Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematop (show more...)Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. We focused on the proteomic characterization of nonmineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic, and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent 5-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to 2-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 253.9 (pelleting) Protein markers EV: Annexin2/ CD63 non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g 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 SW28 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ Annexin2 ELISA Antibody details provided? No Detected EV-associated proteins Annexin2 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140270	2/3	Homo sapiens	NAY	(d)(U)C DG	Menck K	2014	33%
Study summary Full title Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN All authors Menck K, Scharf C, Bleckmann A, Dyck L, Rost U, Wenzel D, Dhople VM, Siam L, Pukrop T, Binder C, Klemm F Journal J Mol Cell Biol Abstract Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are (show more...)Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Tubulin/ TSG101/ MUC1/ Flotillin2/ EMMPRIN non-EV: Proteomics no EV density (g/ml) 1.15-1.18 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 Pelleting performed Yes Pelleting: time(min) 35 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.25 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ EMMPRIN/ MUC1/ Flotillin2/ Tubulin ELISA Antibody details provided? No Detected EV-associated proteins EMMPRIN/ MUC1/ Flotillin2/ Tubulin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field Report size (nm) Not reported

	EV140270	3/3	Homo sapiens	NAY	(d)(U)C DG Filtration	Menck K	2014	33%
Study summary Full title Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN All authors Menck K, Scharf C, Bleckmann A, Dyck L, Rost U, Wenzel D, Dhople VM, Siam L, Pukrop T, Binder C, Klemm F Journal J Mol Cell Biol Abstract Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are (show more...)Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: TSG101/ Flotillin2/ EMMPRIN non-EV: Proteomics yes EV density (g/ml) 1.13-1.22 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) 120 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.25 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 TSG101/ EMMPRIN/ Flotillin2 ELISA Antibody details provided? No Detected EV-associated proteins EMMPRIN/ Flotillin2 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field Report size (nm) Not reported

	EV140115	2/4	Homo sapiens	NAY	(d)(U)C	Lozito TP	2014	33%
Study summary Full title Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors All authors Lozito TP, Tuan RS Journal J Cell Mol Med Abstract Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSC (show more...)Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell-derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non-MP secreted factors (Sup) were isolated from serum-free medium conditioned by human microvascular ECs (HMEC-1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP-containing MPs were isolated from cells transduced with CMV-GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP-MPs, but not free GFP. Thus, only MP-associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP-1, MMP-3, CCL-2/MCP-1 and IL-6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF-?B signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles microparticles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Calnexin/ Caveolin/ GAPDH non-EV: HSP70/ CD63/ LAMP2/ Rab5 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Calnexin/ Caveolin/ GAPDH Detected contaminants "LAMP2/ Rab5/ CD63/ HSP70" ELISA Antibody details provided? No Detected EV-associated proteins Calnexin/ Caveolin/ GAPDH Characterization: Particle analysis None

	EV140115	3/4	Homo sapiens	NAY	(d)(U)C	Lozito TP	2014	33%
Study summary Full title Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors All authors Lozito TP, Tuan RS Journal J Cell Mol Med Abstract Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSC (show more...)Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell-derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non-MP secreted factors (Sup) were isolated from serum-free medium conditioned by human microvascular ECs (HMEC-1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP-containing MPs were isolated from cells transduced with CMV-GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP-MPs, but not free GFP. Thus, only MP-associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP-1, MMP-3, CCL-2/MCP-1 and IL-6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF-?B signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles microparticles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: HSP70/ CD63/ Rab5/ LAMP2/ GAPDH 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 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) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ Rab5/ LAMP2/ GAPDH Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Rab5/ LAMP2/ GAPDH Characterization: Particle analysis None

	EV140048	1/1	Homo sapiens	NAY	(d)(U)C DG Filtration	Lai CP	2014	33%
Study summary Full title Dynamic biodistribution of extracellular vesicles in vivo using a multimodal imaging reporter All authors Lai CP, Mardini O, Ericsson M, Prabhakar S, Maguire CA, Chen JW, Tannous BA, Breakefield XO Journal ACS Nano Abstract Extracellular vesicles (EVs) are nanosized vesicles released by normal and diseased cells as a novel (show more...)Extracellular vesicles (EVs) are nanosized vesicles released by normal and diseased cells as a novel form of intercellular communication and can serve as an effective therapeutic vehicle for genes and drugs. Yet, much remains unknown about the in vivo properties of EVs such as tissue distribution, blood levels, and urine clearance, important parameters that will define their therapeutic effectiveness and potential toxicity. Here we combined Gaussia luciferase and metabolic biotinylation to create a sensitive EV reporter (EV-GlucB) for multimodal imaging in vivo, as well as monitoring of EV levels in the organs and biofluids ex vivo after administration of EVs. Bioluminescence and fluorescence-mediated tomography imaging on mice displayed a predominant localization of intravenously administered EVs in the spleen followed by the liver. Monitoring EV signal in the organs, blood, and urine further revealed that the EVs first undergo a rapid distribution phase followed by a longer elimination phase via hepatic and renal routes within six hours, which are both faster than previously reported using dye-labeled EVs. Moreover, we demonstrate systemically injected EVs can be delivered to tumor sites within an hour following injection. Altogether, we show the EVs are dynamically processed in vivo with accurate spatiotemporal resolution and target a number of normal organs as well as tumors with implications for disease pathology and therapeutic design. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 Protein markers EV: Alix non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 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 8 Highest density fraction 60 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 Alix Characterization: Particle analysis NTA EM EM-type immune EM EM protein CD63 Image type Close-up, Wide-field

	EV140289	3/3	Homo sapiens	Blood plasma	(d)(U)C DG Filtration	Kranendonk ME	2014	33%
Study summary Full title Effect of extracellular vesicles of human adipose tissue on insulin signaling in liver and muscle cells All authors Kranendonk ME, Visseren FL, van Herwaarden JA, Nolte-'t Hoen EN, de Jager W, Wauben MH, Kalkhoven E Journal Obesity Abstract OBJECTIVE: Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To (show more...)OBJECTIVE: Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined. METHODS: EVs released from human subcutaneous (SAT) and omental AT (OAT)-explants ex vivo were used for stimulation of hepatocytes and myotubes in vitro. Subsequently, insulin-induced Akt phosphorylation and expression of gluconeogenic genes (G6P, PEPCK) was determined. AT-EV adipokine levels were measured by multiplex immunoassay, and AT-EVs were quantified by high-resolution flow cytometry. RESULTS: In hepatocytes, AT-EVs from the majority of patients inhibited insulin-induced Akt phosphorylation, while EVs from some patients stimulated insulin-induced Akt phosphorylation. In myotubes AT-EVs exerted an ambiguous effect on insulin signaling. Hepatic Akt phosphorylation related negatively to G6P-expression by both SAT-EVs (r = -0.60, P = 0.01) and OAT-EVs (r = -0.74, P = 0.001). MCP-1, IL-6, and MIF concentrations were higher in OAT-EVs compared to SAT-EVs and differently related to lower Akt phosphorylation in hepatocytes. Finally, the number of OAT-EVs correlated positively with liver enzymes indicative for liver dysfunction. CONCLUSIONS: Human AT-EVs can stimulate or inhibit insulin signaling in hepatocytes- possibly depending on their adipokine content- and may thereby contribute to systemic IR. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 256 (pelleting) Protein markers EV: CD9 non-EV: Proteomics no EV density (g/ml) 1.15-1.2 Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Wash: volume per pellet (ml) 50 Density gradient Lowest density fraction 0.4 Highest density fraction 2.5 Orientation Bottom-up Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9 Characterization: Particle analysis None

	EV140054	2/2	Homo sapiens	Adipose tissue	(d)(U)C DG	Kranendonk ME	2014	33%
Study summary Full title Human adipocyte extracellular vesicles in reciprocal signaling between adipocytes and macrophages All authors Kranendonk ME, Visseren FL, van Balkom BW, Nolte-'t Hoen EN, van Herwaarden JA, de Jager W, Schipper HS, Brenkman AB, Verhaar MC, Wauben MH, Kalkhoven E Journal Obesity Abstract OBJECTIVE: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants (show more...)OBJECTIVE: Extracellular vesicles (EVs) released by human adipocytes or adipose tissue (AT)-explants play a role in the paracrine interaction between adipocytes and macrophages, a key mechanism in AT inflammation, leading to metabolic complications like insulin resistance (IR) were determined. METHODS: EVs released from in vitro differentiated adipocytes and AT-explants ex vivo were characterized by electron microscopy, Western blot, multiplex adipokine-profiling, and quantified by flow cytometry. Primary monocytes were stimulated with EVs from adipocytes, subcutaneous (SCAT) or omental-derived AT (OAT), and phenotyped. Macrophage supernatant was subsequently used to assess the effect on insulin signaling in adipocytes. RESULTS: Adipocyte and AT-derived EVs differentiated monocytes into macrophages characteristic of human adipose tissue macrophages (ATM), defined by release of both pro- and anti-inflammatory cytokines. The adiponectin-positive subset of AT-derived EVs, presumably representing adipocyte-derived EVs, induced a more pronounced ATM-phenotype than the adiponectin-negative AT-EVs. This effect was more evident for OAT-EVs versus SCAT-EVs. Furthermore, supernatant of macrophages pre-stimulated with AT-EVs interfered with insulin signaling in human adipocytes. Finally, the number of OAT-derived EVs correlated positively with patients HOMA-IR. CONCLUSIONS: A possible role for human AT-EVs in a reciprocal pro-inflammatory loop between adipocytes and macrophages, with the potential to aggravate local and systemic IR was demonstrated. (hide) EV-METRIC 33% (41st 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 Adipose tissue 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 256 (pelleting) Protein markers EV: CD9 non-EV: Proteomics no EV density (g/ml) 1.12-1.25 Show all info Study aim Function Sample Species Homo sapiens Sample Type Adipose tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW32;SW40;SW60 Pelleting: adjusted k-factor 256.0 Density gradient Lowest density fraction 0.4 Highest density fraction 2.5 Orientation Bottom-up Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9 Characterization: Particle analysis

	EV140047	1/2	Homo sapiens	NAY	(d)(U)C Filtration	Gonzalez E	2014	33%
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 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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: Flotilin1/ CD63/ CD133/ CD13 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 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/ Flotilin1/ CD133/ CD13 ELISA Antibody details provided? No Detected EV-associated proteins CD133/ CD13 Characterization: Particle analysis NTA EM EM-type cryo EM Image type Wide-field

	EV140112	1/1	Candida albicans	Yeast	(d)(U)C UF	Gil-Bona A	2014	33%
Study summary Full title Proteomics Unravels Extracellular Vesicles as Carriers of Classical Cytoplasmic Proteins in Candida albicans All authors Gil-Bona A, Llama-Palacios A, Parra CM, Vivanco F, Nombela C, Monteoliva L, Gil C Journal J Proteome Res Abstract The commensal fungus Candida albicans secretes a considerable number of proteins and, as in differen (show more...)The commensal fungus Candida albicans secretes a considerable number of proteins and, as in different fungal pathogens, extracellular vesicles (EVs) have also been observed. Our report contains the first proteomic analysis of EVs in C. albicans and a comparative proteomic study of the soluble secreted proteins. With this purpose, cell-free culture supernatants from C. albicans were separated into EVs and EV-free supernatant and analyzed by LC-MS/MS. A total of 96 proteins were identified including 75 and 61 proteins in EVs and EV-free supernatant, respectively. Out of these, 40 proteins were found in secretome by proteomic analysis for the first time. The soluble proteins were enriched in cell wall and secreted pathogenesis related proteins. Interestingly, more than 90% of these EV-free supernatant proteins were classical secretory proteins with predicted N-terminal signal peptide, whereas all the leaderless proteins involved in metabolism, including some moonlighting proteins, or in the exocytosis and endocytosis process were exclusively cargo of the EVs. We propose a model of the different mechanisms used by C. albicans secreted proteins to reach the extracellular medium. Furthermore, we tested the potential of the Bgl2 protein, identified in vesicles and EV-free supernatant, to protect against a systemic candidiasis in a murine model. (hide) EV-METRIC 33% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Yeast Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: BGL2/ TDH3 non-EV: Proteomics yes Show all info Study aim Omics Sample Species Candida albicans Sample Type Yeast Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TDH3/ BGL2 ELISA Antibody details provided? No Detected EV-associated proteins TDH3/ BGL2 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140120	7/7	Homo sapiens	Blood plasma	(d)(U)C Filtration Vn peptides	Ghosh A	2014	33%
Study summary Full title Rapid isolation of extracellular vesicles from cell culture and biological fluids using a synthetic peptide with specific affinity for heat shock proteins All authors Ghosh A, Davey M, Chute IC, Griffiths SG, Lewis S, Chacko S, Barnett D, Crapoulet N, Fournier S, Joy A, Caissie MC, Ferguson AD, Daigle M, Meli MV, Lewis SM, Ouellette RJ Journal PLoS One Abstract Recent studies indicate that extracellular vesicles are an important source material for many clinic (show more...)Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Vn peptides Protein markers EV: non-EV: Proteomics yes 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 Pelleting performed No Filtration steps 0.22µm or 0.2µm Other Name other separation method Vn peptides Characterization: Protein analysis Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140110	1/2	Mus musculus	NAY	(d)(U)C	Cossetti C	2014	33%
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 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 122.2 (pelleting) / 89.21 (washing) Protein markers EV: TSG101/ CD63/ HSP90/ Alix/ Tnfr1/ HSP70/ Beta-actin/ Ago1/ CD9/ Ago2 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 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 EM EM-type transmission EM Image type Wide-field

	EV140240	1/2	Homo sapiens	NAY	(d)(U)C	Zhao X	2014	33%
Study summary Full title Quantitative Proteomic Analysis of Exosome Protein Content Changes Induced by Hepatitis B Virus in Huh-7 Cells Using SILAC Labeling and LC-MS/MS All authors Zhao X, Wu Y, Duan J, Ma Y, Shen Z, Wei L, Cui X, Zhang J, Xie Y, Liu J Journal J Proteome Res Abstract Hepatitis B virus (HBV) infection could cause hepatitis, liver cirrhosis, and hepatocellular carcino (show more...)Hepatitis B virus (HBV) infection could cause hepatitis, liver cirrhosis, and hepatocellular carcinoma. HBV-mediated pathogenesis is only partially understood, but X protein (HBx) reportedly possesses oncogenic potential. Exosomes are small membrane vesicles with diverse functions released by various cells including hepatocytes, and HBV harnesses cellular exosome biogenesis and export machineries for virion morphogenesis and secretion. Therefore, HBV infection might cause changes in exosome contents with functional implications for both virus and host. In this work, exosome protein content changes induced by HBV and HBx were quantitatively analyzed by SILAC/LC-MS/MS. Exosomes prepared from SILAC-labeled hepatoma cell line Huh-7 transfected with HBx, wildtype, or HBx-null HBV replicon plasmids were analyzed by LC-MS/MS. Systematic analyses of MS data and confirmatory immunoblotting showed that HBx overexpression and HBV, with or without HBx, replication in Huh-7 cells indeed caused marked and specific changes in exosome protein contents. Furthermore, specific changes in protein contents were also detected in exosomes purified from HBV-infected patients' sera compared with control sera negative for HBV markers. These results illustrate a new aspect of interactions between HBV and the host and provide the foundation for future research into roles played by exosomes in HBV infection and pathogenesis. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 256 (pelleting) / 255.8 (washing) Protein markers EV: Alix/ TSG101 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Wash: Rotor Type SW41 Wash: adjusted k-factor 255.8 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis None

	EV140240	2/2	Homo sapiens	Serum	(d)(U)C	Zhao X	2014	33%
Study summary Full title Quantitative Proteomic Analysis of Exosome Protein Content Changes Induced by Hepatitis B Virus in Huh-7 Cells Using SILAC Labeling and LC-MS/MS All authors Zhao X, Wu Y, Duan J, Ma Y, Shen Z, Wei L, Cui X, Zhang J, Xie Y, Liu J Journal J Proteome Res Abstract Hepatitis B virus (HBV) infection could cause hepatitis, liver cirrhosis, and hepatocellular carcino (show more...)Hepatitis B virus (HBV) infection could cause hepatitis, liver cirrhosis, and hepatocellular carcinoma. HBV-mediated pathogenesis is only partially understood, but X protein (HBx) reportedly possesses oncogenic potential. Exosomes are small membrane vesicles with diverse functions released by various cells including hepatocytes, and HBV harnesses cellular exosome biogenesis and export machineries for virion morphogenesis and secretion. Therefore, HBV infection might cause changes in exosome contents with functional implications for both virus and host. In this work, exosome protein content changes induced by HBV and HBx were quantitatively analyzed by SILAC/LC-MS/MS. Exosomes prepared from SILAC-labeled hepatoma cell line Huh-7 transfected with HBx, wildtype, or HBx-null HBV replicon plasmids were analyzed by LC-MS/MS. Systematic analyses of MS data and confirmatory immunoblotting showed that HBx overexpression and HBV, with or without HBx, replication in Huh-7 cells indeed caused marked and specific changes in exosome protein contents. Furthermore, specific changes in protein contents were also detected in exosomes purified from HBV-infected patients' sera compared with control sera negative for HBV markers. These results illustrate a new aspect of interactions between HBV and the host and provide the foundation for future research into roles played by exosomes in HBV infection and pathogenesis. (hide) EV-METRIC 33% (76th 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 Adj. k-factor 256 (pelleting) / 255.8 (washing) Protein markers EV: Alix non-EV: Cell organelle protein Proteomics no Show all info Study aim Omics Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Wash: Rotor Type SW41 Wash: adjusted k-factor 255.8 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix Detected contaminants Cell organelle protein Characterization: Particle analysis None

	EV140053	1/2	Homo sapiens	NAY	(d)(U)C Filtration	Ye SB	2014	33%
Study summary Full title Tumor-derived exosomes promote tumor progression and T-cell dysfunction through the regulation of enriched exosomal microRNAs in human nasopharyngeal carcinoma All authors Ye SB, Li ZL, Luo DH, Huang BJ, Chen YS, Zhang XS, Cui J, Zeng YX, Li J Journal Oncotarget Abstract Tumor-derived exosomes contain biologically active proteins and messenger and microRNAs (miRNAs). Th (show more...)Tumor-derived exosomes contain biologically active proteins and messenger and microRNAs (miRNAs). These particles serve as vehicles of intercellular communication and are emerging mediators of tumorigenesis and immune escape. Here, we isolated 30-100 nm exosomes from the serum of patients with nasopharyngeal carcinoma (NPC) or the supernatant of TW03 cells. Increased circulating exosome concentrations were correlated with advanced lymphoid node stage and poor prognosis in NPC patients (P< 0.05). TW03-derived exosomes impaired T-cell function by inhibiting T-cell proliferation and Th1 and Th17 differentiation and promoting Treg induction by NPC cells in vitro. These results are associated with decreases in ERK, STAT1, and STAT3 phosphorylation and increases in STAT5 phosphorylation in exosome-stimulated T-cells. TW03-derived exosomes increased the proinflammatory cytokines IL-1?, IL-6, and IL-10 but decreased IFN?, IL-2, and IL-17 release from CD4+ or CD8+ T-cells. Furthermore, five commonly over-expressed miRNAs were identified in the exosomes from patient sera or NPC cells: hsa-miR-24-3p, hsa-miR-891a, hsa-miR-106a-5p, hsa-miR-20a-5p, and hsa-miR-1908. These over-expressed miRNA clusters down-regulated the MARK1 signaling pathway to alter cell proliferation and differentiation. Overall, these observations reveal the clinical relevance and prognostic value of tumor-derived exosomes and identify a unique intercellular mechanism mediated by tumor-derived exosomes to modulate T-cell function in NPC. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: HSP70/ CD63/ LAMP1/ MHC1 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) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ MHC1/ LAMP1 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins MHC1/ LAMP1 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140234	1/1	Homo sapiens	NAY	(d)(U)C ExoQuick	Xiao X	2014	33%
Study summary Full title Exosomes: decreased sensitivity of lung cancer A549 cells to cisplatin All authors Xiao X, Yu S, Li S, Wu J, Ma R, Cao H, Zhu Y, Feng J Journal PLoS One Abstract Exosomes are small extracellular membrane vesicles of endocytic origin released by many cells that c (show more...)Exosomes are small extracellular membrane vesicles of endocytic origin released by many cells that could be found in most body fluids. The main functions of exosomes are cellular communication and cellular waste clean-up. This study was conducted to determine the involvement of exosomes in the regulation of sensitivity of the lung cancer cell line A549 to cisplatin (DDP). When DDP was added to A549 cells, exosomes secretion was strengthened. Addition of the secreted exosomes to other A549 cells increased the resistance of these A549 cells to DDP. Upon exposure of A549 to DDP, the expression levels of several miRNAs and mRNAs reportedly associated with DDP sensitivity changed significantly in exosomes; these changes may mediate the resistance of A549 cells to DDP. Exosomes released by A549 cells during DDP exposure decreased the sensitivity of other A549 cells to DDP, which may be mediated by miRNAs and mRNAs exchange by exosomes via cell-to-cell communication. Although the detailed mechanism of resistance remains unclear, we believed that inhibition of exosomes formation and release might present a novel strategy for lung cancer treatment in the future. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick Adj. k-factor 156.9 (pelleting) Protein markers EV: CD63 non-EV: Beta-actin Proteomics no TEM measurements 50-70 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) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Detected contaminants Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field Report size (nm) 50-70

	EV140045	1/2	Homo sapiens	NAY	(d)(U)C DC	Webber J	2014	33%
Study summary Full title Proteomics analysis of cancer exosomes using a novel modified aptamer-based array (SOMAscan) platform All authors Webber J, Stone TC, Katilius E, Smith BC, Gordon B, Mason MD, Tabi Z, Brewis IA, Clayton A Journal Mol Cell Proteomics Abstract We have used a novel affinity-based proteomics technology to examine the protein signature of small (show more...)We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes. The technology uses a new class of protein binding reagents called SOMAmers® (slow off-rate modified aptamers) and allows the simultaneous precise measurement of over 1000 proteins. Exosomes were highly purified from the Du145 prostate cancer cell line, by pooling selected fractions from a continuous sucrose gradient (within the density range of 1.1 to 1.2 g/ml), and examined under standard conditions or with additional detergent treatment by the SOMAscan™ array (version 3.0). Lysates of Du145 cells were also prepared, and the profiles were compared. Housekeeping proteins such as cyclophilin-A, LDH, and Hsp70 were present in exosomes, and we identified almost 100 proteins that were enriched in exosomes relative to cells. These included proteins of known association with cancer exosomes such as MFG-E8, integrins, and MET, and also those less widely reported as exosomally associated, such as ROR1 and ITIH4. Several proteins with no previously known exosomal association were confirmed as exosomally expressed in experiments using individual SOMAmer® reagents or antibodies in micro-plate assays. Western blotting confirmed the SOMAscan™-identified enrichment of exosomal NOTCH-3, L1CAM, RAC1, and ADAM9. In conclusion, we describe here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmer®-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Protein markers EV: CD81/ TSG101/ CD63/ CD9/ L1CAM non-EV: Cell organelle protein Proteomics no Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed No Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ TSG101/ L1CAM Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ TSG101/ L1CAM Detected contaminants Cell organelle protein Characterization: Particle analysis None

	EV140042	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Valencia K	2014	33%
Study summary Full title miRNA cargo within exosome-like vesicle transfer influences metastatic bone colonization All authors Valencia K, Luis-Ravelo D, Bovy N, Antón I, Martínez-Canarias S, Zandueta C, Ormazábal C, Struman I, Tabruyn S, Rebmann V, De Las Rivas J, Guruceaga E, Bandrés E, Lecanda F Journal Mol Oncol Abstract Bone metastasis represents one of the most deleterious clinical consequences arising in the context (show more...)Bone metastasis represents one of the most deleterious clinical consequences arising in the context of many solid tumors. Severe osteolysis results from tumor cell colonization of the bone compartment, a process which entails reciprocal exchange of soluble signals between tumor cells and their osseous microenvironment. Recent evidence indicates that tumor-intrinsic miRNAs are pleiotropic regulators of gene expression. But they are also frequently released in exosome-like vesicles (ELV). Yet the functional relevance of the transference of tumor-derived ELV and their miRNA cargo to the extracellular milieu during osseous colonization is unknown. Comparative transcriptomic profiling using an in vivo murine model of bone metastasis identified a repressed miRNA signature associated with high prometastatic activity. Forced expression of single miRNAs identified miR-192 that markedly appeased osseous metastasis in vivo, as shown by X-ray, bioluminescence imaging and microCT scans. Histological examination of metastatic lesions revealed impaired tumor-induced angiogenesis in vivo, an effect that was associated in vitro with decreased hallmarks of angiogenesis. Isolation and characterization of ELV by flow cytometry, Western blot analysis, transmission electron microscopy and nanoparticle tracking analysis revealed the ELV cargo enrichment in miR-192. Consistent with these findings, fluorescent labeled miR-192-enriched-ELV showed the in vitro transfer and release of miR-192 in target endothelial cells and abrogation of the angiogenic program by repression of proangiogenic IL-8, ICAM and CXCL1. Moreover, in vivo infusion of fluorescent labeled ELV efficiently targeted cells of the osseous compartment. Furthermore, treatment with miR-192 enriched ELV in a model of in vivo bone metastasis pre-conditioned osseous milieu and impaired tumor-induced angiogenesis, thereby reducing the metastatic burden and tumor colonization. Changes in the miRNA-cargo content within ELV represent a novel mechanism heavily influencing bone metastatic colonization, which is most likely relevant in other target organs. Mechanistic mimicry of this phenomenon by synthetic nanoparticles could eventually emerge as a novel therapeutic approach. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Exosome-like vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: Alix/ TSG101/ 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) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140284	2/2	Homo sapiens	NAY	(d)(U)C Filtration Microfluidics Total Exosome Isolation	Vaidyanathan R	2014	33%
Study summary Full title Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing All authors Vaidyanathan R, Naghibosadat M, Rauf S, Korbie D, Carrascosa LG, Shiddiky MJ, Trau M Journal Anal Chem Abstract Exosomes show promise as noninvasive biomarkers for cancer, but their effective capture and specific (show more...)Exosomes show promise as noninvasive biomarkers for cancer, but their effective capture and specific detection is a significant challenge. Herein, we report a multiplexed microfluidic device for highly specific capture and detection of multiple exosome targets using a tunable alternating current electrohydrodynamic (ac-EHD) methodology, referred to as nanoshearing. In our system, electrical body forces generated by ac-EHD act within nanometers of an electrode surface (i.e., within the electrical layer) to generate nanoscaled fluid flow that enhances the specificity of capture and also reduce nonspecific adsorption of weakly bound molecules from the electrode surface. This approach demonstrates the analysis of exosomes derived from cells expressing human epidermal growth factor receptor 2 (HER2) and prostate specific antigen (PSA), and is also capable of specifically isolating exosomes from breast cancer patient samples. The device also exhibited a 3-fold enhancement in detection sensitivity in comparison to hydrodynamic flow based assays (LOD 2760 exosomes/?L for ac-EHD vs LOD 8300 exosomes/?L for hydrodynamic flow; (n = 3)). We propose this approach can potentially have relevance as a simple and rapid quantification tool to analyze exosome targets in biological applications. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Microfluidics Total Exosome Isolation Protein markers EV: HER2/ PSA/ CD9 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit Total Exosome Isolation Other Name other separation method Microfluidics Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HER2/ PSA ELISA Antibody details provided? No Detected EV-associated proteins CD9/ HER2/ PSA Characterization: Particle analysis DLS TRPS EM EM-type cryo EM Image type Close-up

	EV140221	1/2	Homo sapiens	NAY	(d)(U)C	Srikanthan S	2014	33%
Study summary Full title Exosome poly-ubiquitin inhibits platelet activation, downregulates CD36 and inhibits pro-atherothombotic cellular functions All authors Srikanthan S, Li W, Silverstein RL, McIntyre TM Journal J Thromb Haemost Abstract INTRODUCTION: Activated platelets shed microparticles from plasma membranes, but also release smalle (show more...)INTRODUCTION: Activated platelets shed microparticles from plasma membranes, but also release smaller exosomes from internal compartments. While microparticles participate in athero-thrombosis, little is known of exosomes in this process. MATERIALS & METHODS: Ex vivo biochemical experiments with human platelets and exosomes, and FeCl3 -induced murine carotid artery thrombosis. RESULTS: Both microparticles and exosomes were abundant in human plasma. Platelet-derived exosomes suppressed ex vivo platelet aggregation and reduced adhesion to collagen-coated microfluidic channels at high shear. Injected exosomes inhibited occlusive thrombosis in FeCl3 -damaged murine carotid arteries. Control platelets infused into irradiated, thrombocytopenic mice reconstituted thrombosis in damaged carotid arteries, but failed to do so after prior ex vivo incubation with exosomes.CD36 promotes platelet activation, and exosomes dramatically reduced platelet CD36.CD36 is also expressed by macrophages, where it binds and internalizes oxidized LDL and microparticles, supplying lipid to promote foam cell formation. Platelet exosomes inhibited oxidized-LDL binding and cholesterol loading into macrophages. Exosomes were not competitive CD36 ligands, but instead sharply reduced total macrophage CD36 content. Exosomal proteins, in contrast to microparticle or cellular proteins, were highly adducted by ubiquitin. Exosomes enhanced ubiquitination of cellular proteins, including CD36, and blockade of proteosome proteolysis with MG-132 rescued CD36 expression. Recombinant unanchored K48 poly-ubiquitin behaved similarly to exosomes, inhibiting platelet function, macrophage CD36 expression and macrophage particle uptake. CONCLUSIONS: Platelet-derived exosomes inhibit athero-thrombotic processes by reducing CD36-dependent lipid loading of macrophages and by suppressing platelet thrombosis. Exosomes increase protein ubiquitination and enhance proteasome degradation of CD36. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ CD9 non-EV: CD36 Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 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 CD63/ CD9 Detected contaminants CD36 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140221	2/2	Homo sapiens	Platelet supernatant	(d)(U)C	Srikanthan S	2014	33%
Study summary Full title Exosome poly-ubiquitin inhibits platelet activation, downregulates CD36 and inhibits pro-atherothombotic cellular functions All authors Srikanthan S, Li W, Silverstein RL, McIntyre TM Journal J Thromb Haemost Abstract INTRODUCTION: Activated platelets shed microparticles from plasma membranes, but also release smalle (show more...)INTRODUCTION: Activated platelets shed microparticles from plasma membranes, but also release smaller exosomes from internal compartments. While microparticles participate in athero-thrombosis, little is known of exosomes in this process. MATERIALS & METHODS: Ex vivo biochemical experiments with human platelets and exosomes, and FeCl3 -induced murine carotid artery thrombosis. RESULTS: Both microparticles and exosomes were abundant in human plasma. Platelet-derived exosomes suppressed ex vivo platelet aggregation and reduced adhesion to collagen-coated microfluidic channels at high shear. Injected exosomes inhibited occlusive thrombosis in FeCl3 -damaged murine carotid arteries. Control platelets infused into irradiated, thrombocytopenic mice reconstituted thrombosis in damaged carotid arteries, but failed to do so after prior ex vivo incubation with exosomes.CD36 promotes platelet activation, and exosomes dramatically reduced platelet CD36.CD36 is also expressed by macrophages, where it binds and internalizes oxidized LDL and microparticles, supplying lipid to promote foam cell formation. Platelet exosomes inhibited oxidized-LDL binding and cholesterol loading into macrophages. Exosomes were not competitive CD36 ligands, but instead sharply reduced total macrophage CD36 content. Exosomal proteins, in contrast to microparticle or cellular proteins, were highly adducted by ubiquitin. Exosomes enhanced ubiquitination of cellular proteins, including CD36, and blockade of proteosome proteolysis with MG-132 rescued CD36 expression. Recombinant unanchored K48 poly-ubiquitin behaved similarly to exosomes, inhibiting platelet function, macrophage CD36 expression and macrophage particle uptake. CONCLUSIONS: Platelet-derived exosomes inhibit athero-thrombotic processes by reducing CD36-dependent lipid loading of macrophages and by suppressing platelet thrombosis. Exosomes increase protein ubiquitination and enhance proteasome degradation of CD36. (hide) EV-METRIC 33% (83rd 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 Platelet 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/ CD9 non-EV: CD36 Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Platelet supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD9 Detected contaminants CD36 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140220	1/2	Mus musculus	NAY	(d)(U)C	Squadrito ML	2014	33%
Study summary Full title Endogenous RNAs modulate microRNA sorting to exosomes and transfer to acceptor cells All authors Squadrito ML, Baer C, Burdet F, Maderna C, Gilfillan GD, Lyle R, Ibberson M, De Palma M Journal Cell Rep Abstract MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specif (show more...)MicroRNA (miRNA) transfer via exosomes may mediate cell-to-cell communication. Interestingly, specific miRNAs are enriched in exosomes in a cell-type-dependent fashion. However, the mechanisms whereby miRNAs are sorted to exosomes and the significance of miRNA transfer to acceptor cells are unclear. We used macrophages and endothelial cells (ECs) as a model of heterotypic cell communication in order to investigate both processes. RNA profiling of macrophages and their exosomes shows that miRNA sorting to exosomes is modulated by cell-activation-dependent changes of miRNA target levels in the producer cells. Genetically perturbing the expression of individual miRNAs or their targeted transcripts promotes bidirectional miRNA relocation from the cell cytoplasm/P bodies (sites of miRNA activity) to multivesicular bodies (sites of exosome biogenesis) and controls miRNA sorting to exosomes. Furthermore, the use of Dicer-deficient cells and reporter lentiviral vectors (LVs) for miRNA activity shows that exosomal miRNAs are transferred from macrophages to ECs to detectably repress targeted sequences. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ CD81/ CD9 non-EV: GAPDH Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81/ CD9 Detected contaminants GAPDH Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV140098	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Shimoda M	2014	33%
Study summary Full title Loss of the Timp gene family is sufficient for the acquisition of the CAF-like cell state All authors Shimoda M, Principe S, Jackson HW, Luga V, Fang H, Molyneux SD, Shao YW, Aiken A, Waterhouse PD, Karamboulas C, Hess FM, Ohtsuka T, Okada Y, Ailles L, Ludwig A, Wrana JL, Kislinger T, Khokha R Journal Nat Cell Biol Abstract Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state (show more...)Cancer-associated fibroblasts (CAFs) drive tumour progression, but the emergence of this cell state is poorly understood. A broad spectrum of metalloproteinases, controlled by the Timp gene family, influence the tumour microenvironment in human cancers. Here, we generate quadruple TIMP knockout (TIMPless) fibroblasts to unleash metalloproteinase activity within the tumour-stromal compartment and show that complete Timp loss is sufficient for the acquisition of hallmark CAF functions. Exosomes produced by TIMPless fibroblasts induce cancer cell motility and cancer stem cell markers. The proteome of these exosomes is enriched in extracellular matrix proteins and the metalloproteinase ADAM10. Exosomal ADAM10 increases aldehyde dehydrogenase expression in breast cancer cells through Notch receptor activation and enhances motility through the GTPase RhoA. Moreover, ADAM10 knockdown in TIMPless fibroblasts abrogates their CAF function. Importantly, human CAFs secrete ADAM10-rich exosomes that promote cell motility and activate RhoA and Notch signalling in cancer cells. Thus, Timps suppress cancer stroma where activated-fibroblast-secreted exosomes impact tumour progression. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: ADAM10/ CD81/ Alpha/beta-tubulin/ Flotilin1 non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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) 120-480 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/ Flotilin1/ ADAM10/ Alpha/beta-tubulin ELISA Antibody details provided? No Detected EV-associated proteins ADAM10/ Alpha/beta-tubulin Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein ADAM10 Image type Close-up, Wide-field

	EV140096	1/1	Bos bovis	FBS	(d)(U)C Filtration	Shelke GV	2014	33%
Study summary Full title Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum All authors Shelke GV, Lässer C, Gho YS, Lötvall J Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multi (show more...)Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type FBS 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 Adj. k-factor 174.8 (pelleting) / 174.8 (washing) Protein markers EV: CD81/ TSG101/ CD63 non-EV: Proteomics no Show all info Study aim Technical Sample Species Bos bovis Sample Type FBS Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 960 Pelleting: rotor type 45Ti Pelleting: adjusted k-factor 174.8 Wash: Rotor Type 45Ti Wash: adjusted k-factor 174.8 Filtration steps 0.22µm or 0.2µm0.2µm > x > 0.1µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ TSG101 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140216	1/1	Homo sapiens	Urine	(d)(U)C UF	Saraswat M	2014	33%
Study summary Full title N-glycoproteomics of urinary exosomes All authors Saraswat M, Joenväära S, Musante L, Peltoniemi H, Holthofer H, Renkonen R Journal Mol Cell Proteomics Abstract Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted (show more...)Epithelial cells lining the urinary tract secrete urinary exosomes (40-100 nm) that can be targeted to specific cells modulating their functionality. One potential targeting mechanism is adhesion between vesicle surface glycoproteins and target cells. This makes the glycopeptide analysis of exosomes important. Exosomes reflect the physiological state of the parent cells; therefore, they are a good source of biomarkers for urological and other diseases. Moreover, the urine collection is easy and noninvasive and urinary exosomes give information about renal and systemic organ systems. Accordingly, multiple studies on proteomic characterization of urinary exosomes in health and disease have been published. However, no systematic analysis of their glycoproteomic profile has been carried out to date, whereas a conserved glycan signature has been found for exosomes from urine and other sources including T cell lines and human milk. Here, we have enriched and identified the N-glycopeptides from these vesicles. These enriched N-glycopeptides were solved for their peptide sequence, glycan composition, structure, and glycosylation site using collision-induced dissociation MS/MS (CID-tandem MS) data interpreted by a publicly available software GlycopeptideId. Released glycans from the same sample was also analyzed with MALDI-MS. We have identified the N-glycoproteome of urinary exosomes. In total 126 N-glycopeptides from 51 N-glycosylation sites belonging to 37 glycoproteins were found in our results. The peptide sequences of these N-glycopeptides were identified unambiguously and their glycan composition (for 125 N-glycopeptides) and structures (for 87 N-glycopeptides) were proposed. A corresponding glycomic analysis with released N-glycans was also performed. We identified 66 unique nonmodified N-glycan compositions and in addition 13 sulfated/phosphorylated glycans were also found. This is the first systematic analysis of N-glycoproteome of urinary exosomes. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Adj. k-factor 61.11 (pelleting) Protein markers EV: TSG101 non-EV: Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 61.11 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140214	1/1	Homo sapiens	NAY	(d)(U)C DC Filtration	Salomon C	2014	33%
Study summary Full title Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration All authors Salomon C, Yee S, Scholz-Romero K, Kobayashi M, Vaswani K, Kvaskoff D, Illanes SE, Mitchell MD, Rice GE Journal Front Pharmacol Abstract BACKGROUND: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterin (show more...)BACKGROUND: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration. METHODS: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37°C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected, and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software). RESULTS: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 × 10(8) ± 2.5 × 10(8) particles/10(6) cells) compared to JEG-3 (2.86 × 10(8) ± 0.78 × 10(8) particles/10(6) cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (-exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, vs. control 25.09 ± 0.58 h, p < 0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes. CONCLUSION: The data obtained in this study are consistent with the hypothesis that the release, content, and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration Adj. k-factor 221.9 (pelleting) Protein markers EV: CD63 non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 75 Pelleting: rotor type SureSpin630 Pelleting: adjusted k-factor 221.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63 ELISA Antibody details provided? No Detected EV-associated proteins CD63 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140210	1/1	Equus caballus	Serum	(d)(U)C DG	Rout ED	2014	33%
Study summary Full title Transferrin receptor expression in serum exosomes as a marker of regenerative anaemia in the horse All authors Rout ED, Webb TL, Laurence HM, Long L, Olver CS Journal Equine Vet J Abstract REASONS FOR PERFORMING STUDY: Evaluation of erythrocyte regeneration in horses is challenging, as th (show more...)REASONS FOR PERFORMING STUDY: Evaluation of erythrocyte regeneration in horses is challenging, as they do not release reticulocytes into the peripheral blood. This study investigated transferrin receptor 1 (TfR1) expression in exosomes as a noninvasive method of characterising the regenerative response in anaemic horses. OBJECTIVES: To quantify TfR1 in ultraprecipitate of serum in horses before and after phlebotomy-induced anaemia, and to identify exosomes as the source of TfR1. The hypothesis was that serum exosomal TfR1 expression would increase during a regenerative response. STUDY DESIGN: Experimental model of anaemia. METHODS: Six horses were phlebotomised to achieve a 25% decrease in packed cell volume. Transferrin receptor 1 quantity in exosomes was determined by western blot and relative densitometry before and after phlebotomy. The size and density of the TfR1-associated particles were confirmed by transmission electron microscopy and density gradient centrifugation, respectively. RESULTS: Regenerative anaemia was confirmed by decreased packed cell volumes and decreased myeloid:erythroid ratios in the bone marrow. In all 6 horses, TfR1 expression increased between Days 7 and 10. Mean TfR1 levels peaked on Day 10 and at 3-fold higher than levels on Day 0. Appropriately sized particles were evident on transmission electron microscopy and sucrose density gradient fractions expected to contain exosomes also contained TfR1. CONCLUSIONS: These data indicate that TfR1 expression in serum exosomes may provide a marker for regeneration in anaemic horses. (hide) EV-METRIC 33% (76th 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 DG Adj. k-factor 255.8 (pelleting) Protein markers EV: Tf-receptor non-EV: Proteomics no EV density (g/ml) 1.125-1.16 Show all info Study aim Biomarker Sample Species Equus caballus Sample Type Serum 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 SW41 Pelleting: adjusted k-factor 255.8 Wash: volume per pellet (ml) 1 Density gradient Only used for validation of main results Yes Highest density fraction 60 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Tf-receptor ELISA Antibody details provided? No Detected EV-associated proteins Tf-receptor Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140132	2/4	Mus musculus	Blood plasma	(d)(U)C	Povero D	2014	33%
Study summary Full title Circulating Extracellular Vesicles with Specific Proteome and Liver MicroRNAs Are Potential Biomarkers for Liver Injury in Experimental Fatty Liver Disease All authors Povero D, Eguchi A, Li H, Johnson CD, Papouchado BG, Wree A, Messer K, Feldstein AE Journal PLoS One Abstract BACKGROUND & AIM: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease (show more...)BACKGROUND & AIM: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy. DESIGN: Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens. RESULTS: We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver. CONCLUSIONS: These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 1279 (pelleting) Protein markers EV: CD63/ CD81/ ASGPR1/ Annexin5/ ICAM1/ Vanin non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Mus musculus Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 1279. Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ ICAM1/ Annexin5/ Vanin/ ASGPR1 ELISA Antibody details provided? No Detected EV-associated proteins ICAM1/ Annexin5/ Vanin/ ASGPR1 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

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