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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV110097	2/2	Staphylococcus aureus	Skin lavage fluids	(d)(U)C Filtration UF	Hong SW	2011	0%
Study summary Full title Extracellular vesicles derived from Staphylococcus aureus induce atopic dermatitis-like skin inflammation All authors Hong SW, Kim MR, Lee EY, Kim JH, Kim YS, Jeon SG, Yang JM, Lee BJ, Pyun BY, Gho YS, Kim YK Journal Allergy Abstract BACKGROUND: Recently, we found that Staphylococcus aureus produces extracellular vesicles (EV) that (show more...)BACKGROUND: Recently, we found that Staphylococcus aureus produces extracellular vesicles (EV) that contain pathogenic proteins. Although S. aureus infection has been linked with atopic dermatitis (AD), the identities of the causative agents from S. aureus are controversial. We evaluated whether S. aureus-derived EV are causally related to the pathogenesis of AD. METHODS: Extracellular vesicles were isolated by the ultracentrifugation of S. aureus culture media. The EV were applied three times per week to tape-stripped mouse skin. Inflammation and immune dysfunction were evaluated 48 h after the final application in hairless mice. Extracellular vesicles-specific IgE levels were measured by ELISA in AD patients and healthy subjects. RESULTS: The in vitro application of S. aureus EV increased the production of pro-inflammatory mediators (IL-6, thymic stromal lymphopoietin, macrophage inflammatory protein-1?, and eotaxin) by dermal fibroblasts. The in vivo application of S. aureus EV after tape stripping caused epidermal thickening with infiltration of the dermis by mast cells and eosinophils in mice. These changes were associated with the enhanced cutaneous production of IL-4, IL-5, IFN-?, and IL-17. Interestingly, the serum levels of S. aureus EV-specific IgE were significantly increased in AD patients relative to healthy subjects. CONCLUSION: These results indicate that S. aureus EV induce AD-like inflammation in the skin and that S. aureus-derived EV are a novel diagnostic and therapeutic target for the control of AD. (hide) EV-METRIC 0% (median: 0% 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 Skin lavage fluids Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Staphylococcus aureus Sample Type Skin lavage fluids 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) 180 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm

	EV110094	1/1	Homo sapiens Mus musculus	NAY	(d)(U)C ExoQuick Filtration	Hartman ZC	2011	0%
Study summary Full title Increasing vaccine potency through exosome antigen targeting All authors Hartman ZC, Wei J, Glass OK, Guo H, Lei G, Yang XY, Osada T, Hobeika A, Delcayre A, Le Pecq JB, Morse MA, Clay TM, Lyerly HK Journal Vaccine Abstract While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develo (show more...)While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develop efficient TAA cancer vaccines using classical vaccine approaches have been largely ineffective. Recently, a process to specifically target proteins to exosomes has been established which takes advantage of the ability of the factor V like C1C2 domain of lactadherin to specifically address proteins to exosomes. Using this approach, we hypothesized that TAAs could be targeted to exosomes to potentially increase their immunogenicity, as exosomes have been demonstrated to traffic to antigen presenting cells (APC). To investigate this possibility, we created adenoviral vectors expressing the extracellular domain (ECD) of two non-mutated TAAs often found in tumors of cancer patients, carcinoembryonic antigen (CEA) and HER2, and coupled them to the C1C2 domain of lactadherin. We found that these C1C2 fusion proteins had enhanced expression in exosomes in vitro. We saw significant improvement in antigen specific immune responses to each of these antigens in naïve and tolerant transgenic animal models and could further demonstrate significantly enhanced therapeutic anti-tumor effects in a human HER2+ transgenic animal model. These findings demonstrate that the mode of secretion and trafficking can influence the immunogenicity of different human TAAs, and may explain the lack of immunogenicity of non-mutated TAAs found in cancer patients. They suggest that exosomal targeting could enhance future anti-tumor vaccination protocols. This targeting exosome process could also be adapted for the development of more potent vaccines in some viral and parasitic diseases where the classical vaccine approach has demonstrated limitations. (hide) EV-METRIC 0% (median: 14% 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 Filtration Protein markers EV: CD81/ TSG101 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.45µm > x > 0.22µm, Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ TSG101 ELISA Antibody details provided? No Detected EV-associated proteins CD81/ TSG101

	EV110077	1/3	Homo sapiens	Blood plasma	(d)(U)C	Grant R	2011	0%
Study summary Full title A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma All authors Grant R, Ansa-Addo E, Stratton D, Antwi-Baffour S, Jorfi S, Kholia S, Krige L, Lange S, Inal J Journal J Immunol Methods Abstract The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably (show more...)The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 ?m in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 ?m pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 ?m) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 ?M) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects. (hide) EV-METRIC 0% (median: 22% 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 / "Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ Annexin5 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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) 60 Western Blot Antibody details provided? No Detected EV-associated proteins Annexin5 ELISA Antibody details provided? No Detected EV-associated proteins Annexin5

	EV110077	2/3	Homo sapiens	Blood plasma	(d)(U)C	Grant R	2011	0%
Study summary Full title A filtration-based protocol to isolate human plasma membrane-derived vesicles and exosomes from blood plasma All authors Grant R, Ansa-Addo E, Stratton D, Antwi-Baffour S, Jorfi S, Kholia S, Krige L, Lange S, Inal J Journal J Immunol Methods Abstract The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably (show more...)The methods of Plasma Membrane-derived Vesicle (PMV) isolation and quantification vary considerably in the literature and a new standard needs to be defined. This study describes a novel filtration method to isolate PMVs in plasma, which avoids high speed centrifugation, and to quantify them using a Becton Dickinson (BD) FACS Calibur™ flow cytometer, as annexin V-positive vesicles, larger than 0.2 ?m in diameter. Essentially microvesicles (which comprise a mixture of PMVs and exosomes) from citrate plasma were sonicated to break up clumped exosomes, and filtered using Millipore 0.1 ?m pore size Hydrophilic Durapore membranes in Swinnex 13 mm filter holders. Phosphatidylserine-positive PMVs detected with annexin V-PE were quantified using combined labelling and gating strategies in conjunction with Polysciences Polybead Microspheres (0.2 ?m) and BDTrucount tubes. The PMV absolute count was calculated on the analysis template using the Trucount tube lot number information and expressed in PMV count/ml. Having estimated a normal reference range (0.51×10(5)-2.82×10(5) PMVs/ml) from a small sample of human donors, using the developed method, the effect of certain variables was investigated. Variations such as freezing of samples and gender status did not significantly alter the PMV absolute count, and with age plasma PMV levels were only marginally reduced. Smokers appeared to have reduced PMV levels. Nicotine, as for calpeptin was shown to dose-dependently (from 10 up to 50 ?M) reduce levels of early apoptosis in THP-1 monocytes and to decrease the level of PMV release. Fasting individuals had 2-3 fold higher PMV absolute counts compared to non-fasting subjects. (hide) EV-METRIC 0% (median: 22% 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 / "Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ Annexin5 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Western Blot Antibody details provided? No Detected EV-associated proteins Annexin5 ELISA Antibody details provided? No Detected EV-associated proteins Annexin5

	EV110038	1/3	Rattus norvegicus/rattus	NAY	(d)(U)C	Fitzner D	2011	0%
Study summary Full title Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis All authors Fitzner D, Schnaars M, van Rossum D, Krishnamoorthy G, Dibaj P, Bakhti M, Regen T, Hanisch UK, Simons M Journal J Cell Sci Abstract The transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenes (show more...)The transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenesis of autoimmune diseases. Here, we show that oligodendrocytes secrete small membrane vesicles called exosomes, which are specifically and efficiently taken up by microglia both in vitro and in vivo. Internalisation of exosomes occurs by a macropinocytotic mechanism without inducing a concomitant inflammatory response. After stimulation of microglia with interferon-?, we observe an upregulation of MHC class II in a subpopulation of microglia. However, exosomes are preferentially internalised in microglia that do not seem to have antigen-presenting capacity. We propose that the constitutive macropinocytotic clearance of exosomes by a subset of microglia represents an important mechanism through which microglia participate in the degradation of oligodendroglial membrane in an immunologically 'silent' manner. By designating the capacity for macropinocytosis and antigen presentation to distinct cells, degradation and immune function might be assigned to different subtypes of microglia. (hide) EV-METRIC 0% (median: 14% 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: Phosphatidylserine non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Rattus norvegicus/rattus 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 Western Blot Antibody details provided? No Detected EV-associated proteins Phosphatidylserine ELISA Antibody details provided? No Detected EV-associated proteins Phosphatidylserine Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis EM EM-type immune EM EM protein Phosphatidylserine Image type Close-up

	EV110036	2/2	Homo sapiens	Blood plasma	(d)(U)C	Dragovic RA	2011	0%
Study summary Full title Sizing and phenotyping of cellular vesicles using Nanoparticle Tracking Analysis All authors Dragovic RA, Gardiner C, Brooks AS, Tannetta DS, Ferguson DJ, Hole P, Carr B, Redman CW, Harris AL, Dobson PJ, Harrison P, Sargent IL Journal Nanomedicine Abstract Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have m (show more...)Cellular microvesicles and nanovesicles (exosomes) are involved in many disease processes and have major potential as biomarkers. However, developments in this area are constrained by limitations in the technology available for their measurement. Here we report on the use of fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles. In this system vesicles are visualized by light scattering using a light microscope. A video is taken, and the NTA software tracks the brownian motion of individual vesicles and calculates their size and total concentration. Using human placental vesicles and plasma, we have demonstrated that NTA can measure cellular vesicles as small as ? 50 nm and is far more sensitive than conventional flow cytometry (lower limit ? 300 nm). By combining NTA with fluorescence measurement we have demonstrated that vesicles can be labeled with specific antibody-conjugated quantum dots, allowing their phenotype to be determined.FROM THE CLINICAL EDITOR: The authors of this study utilized fluorescence nanoparticle tracking analysis (NTA) to rapidly size and phenotype cellular vesicles, demonstrating that NTA is far more sensitive than conventional flow cytometry. (hide) EV-METRIC 0% (median: 22% 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 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 Protein markers EV: non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Particle analysis NTA

	EV110090	1/1	Homo sapiens	NAY	(d)(U)C	Cullinane AR	2011	0%
Study summary Full title Homozygosity mapping and whole-exome sequencing to detect SLC45A2 and G6PC3 mutations in a single patient with oculocutaneous albinism and neutropenia All authors Cullinane AR, Vilboux T, O'Brien K, Curry JA, Maynard DM, Carlson-Donohoe H, Ciccone C; NISC Comparative Sequencing Program, Markello TC, Gunay-Aygun M, Huizing M, Gahl WA Journal J Invest Dermatol Abstract We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropeni (show more...)We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2. This was ruled out because of the presence of platelet ?-granules and absence of AP3B1 mutations. As parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole-exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional dideoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with OCA type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient's melanocytes caused the mislocalization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient's G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under endoplasmic reticulum stress. To our knowledge, this report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping, followed by whole-exome sequencing, to identify disease-causing mutations in multiple genes in a single affected individual. (hide) EV-METRIC 0% (median: 14% 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 Protein markers EV: TSG101/ Beta-actin non-EV: Proteomics no Show all info Study aim Other/identification of disease causing genes (OCA-4/oculocutaneous albinism type 4 ) 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis None

	EV110089	1/1	Homo sapiens	Serum	(d)(U)C UF	Cubedo J	2011	0%
Study summary Full title Proteomic signature of Apolipoprotein J in the early phase of new-onset myocardial infarction All authors Cubedo J, Padró T, García-Moll X, Pintó X, Cinca J, Badimon L Journal J Proteome Res Abstract Acute myocardial infarction (AMI) is one of the major causes of mortality and morbidity worldwide. D (show more...)Acute myocardial infarction (AMI) is one of the major causes of mortality and morbidity worldwide. Despite all the efforts, there is a lack of early markers for prevention, diagnosis, and treatment of ischemic syndromes. By applying a proteomic expression profiling approach to identify biomarkers of early stages of AMI, we have detected significant changes in Apolipoprotein J/clusterin (ApoJ) in patients with an acute new-onset myocardial infarction. ApoJ characterization by bidimensional electrophoresis (2-DE), followed by mass spectrometry (MALDI-TOF) depicted a cluster of 13 spots (pI, 4.5-5.0; M(w), 37.1-47.3 kDa) with a significantly different distribution between AMI-patients and controls. Specifically, spots 2, 3, 7, 10, and 13 showed a 2-fold increase in their intensity in AMI-patients (P = 0.001). Western-blot analysis (WB) for total serum ApoJ depicted two bands of 40-45 and 65-70 kDa. When only glycosylated forms were analyzed, the band of 65-70 kDa was the most predominant one. A 25% decrease (P = 0.05) of ApoJ glycosylated forms in AMI-patients was detected by 2-DE. Serum ApoJ levels, determined by a commercial ELISA, were significantly lower (P < 0.001) in AMI-patients (n = 39) immediately after the event than in controls (n = 60). In 60% of patients, the lowest ApoJ level was detected within 6 h after the onset of AMI. Between 72 and 96 h after admission, ApoJ values in AMI-patients had reached control levels. Our results demonstrate alterations in ApoJ proteomic profile, due to a differential glycosylation pattern, in AMI-patients within the first 6 h after the onset of the event. Therefore, the analysis of this isoform glycosylation shift in patients with AMI may be of better use to understand ApoJ function than the total serum levels of ApoJ and this isoform shift may become an early marker of AMI. (hide) EV-METRIC 0% (median: 13% 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 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: non-EV: Proteomics no Show all info Study aim Other/Presence of ApoJ soluble or vesicle-associated in serum Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 45 Characterization: Particle analysis None

	EV110081	1/3	Homo sapiens	NAY	(d)(U)C DC IAF	Clayton A	2011	0%
Study summary Full title Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production All authors Clayton A, Al-Taei S, Webber J, Mason MD, Tabi Z Journal J Immunol Abstract Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune respo (show more...)Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 5'AMP-phosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosome-generated adenosine can trigger a cAMP response in adenosine A(2A) receptor-positive but not A(2A) receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28 cross-linking could be inhibited by exogenously added 5'AMP in a CD73-dependent manner. However, 5'AMP converted to adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated through the adenosine A(2A) receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment. (hide) EV-METRIC 0% (median: 14% 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 IAF Protein markers EV: TSG101/ CD39 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 Pelleting performed No Immunoaffinity capture Selected surface protein(s) CD9, CD39, CD73 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ CD39 ELISA Antibody details provided? No Detected EV-associated proteins CD39 Characterization: Particle analysis None

	EV110081	2/3	Homo sapiens	Pleural fluid	(d)(U)C DC	Clayton A	2011	0%
Study summary Full title Cancer exosomes express CD39 and CD73, which suppress T cells through adenosine production All authors Clayton A, Al-Taei S, Webber J, Mason MD, Tabi Z Journal J Immunol Abstract Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune respo (show more...)Extracellular adenosine is elevated in cancer tissue, and it negatively regulates local immune responses. Adenosine production from extracellular ATP has attracted attention as a mechanism of regulatory T cell-mediated immune regulation. In this study, we examined whether small vesicles secreted by cancer cells, called exosomes, contribute to extracellular adenosine production and hence modulate immune effector cells indirectly. We found exosomes from diverse cancer cell types exhibit potent ATP- and 5'AMP-phosphohydrolytic activity, partly attributed to exosomally expressed CD39 and CD73, respectively. Comparable levels of activity were seen with exosomes from pleural effusions of mesothelioma patients. In such fluids, exosomes accounted for 20% of the total ATP-hydrolytic activity. Exosomes can perform both hydrolytic steps sequentially to form adenosine from ATP. This exosome-generated adenosine can trigger a cAMP response in adenosine A(2A) receptor-positive but not A(2A) receptor-negative cells. Similarly, significantly elevated cAMP was also triggered in Jurkat cells by adding exosomes with ATP but not by adding exosomes or ATP alone. A proportion of healthy donor T cells constitutively express CD39 and/or CD73. Activation of T cells by CD3/CD28 cross-linking could be inhibited by exogenously added 5'AMP in a CD73-dependent manner. However, 5'AMP converted to adenosine by exosomes inhibits T cell activation independently of T cell CD73 expression. This T cell inhibition was mediated through the adenosine A(2A) receptor. In summary, the data highlight exosome enzymic activity in the production of extracellular adenosine, and this may play a contributory role in negative modulation of T cells in the tumor environment. (hide) EV-METRIC 0% (median: 25% 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 Pleural fluid 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: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Pleural fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Pelleting performed No Characterization: Particle analysis None

	EV110088	1/1	Homo sapiens	NAY	(d)(U)C Filtration HPLC	Chen TS	2011	0%
Study summary Full title Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs All authors Chen TS, Arslan F, Yin Y, Tan SS, Lai RC, Choo AB, Padmanabhan J, Lee CN, de Kleijn DP, Lim SK Journal J Transl Med Abstract BACKGROUND: Exosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hE (show more...)BACKGROUND: Exosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hESC-MSCs) have been shown to reduce myocardial ischemia/reperfusion injury in animal models. However, as hESC-MSCs are not infinitely expansible, large scale production of these exosomes would require replenishment of hESC-MSC through derivation from hESCs and incur recurring costs for testing and validation of each new batch. Our aim was therefore to investigate if MYC immortalization of hESC-MSC would circumvent this constraint without compromising the production of therapeutically efficacious exosomes. METHODS: The hESC-MSCs were transfected by lentivirus carrying a MYC gene. The transformed cells were analyzed for MYC transgene integration, transcript and protein levels, and surface markers, rate of cell cycling, telomerase activity, karyotype, genome-wide gene expression and differentiation potential. The exosomes were isolated by HPLC fractionation and tested in a mouse model of myocardial ischemia/reperfusion injury, and infarct sizes were further assessed by using Evans' blue dye injection and TTC staining. RESULTS: MYC-transformed MSCs largely resembled the parental hESC-MSCs with major differences being reduced plastic adherence, faster growth, failure to senesce, increased MYC protein expression, and loss of in vitro adipogenic potential that technically rendered the transformed cells as non-MSCs. Unexpectedly, exosomes from MYC-transformed MSCs were able to reduce relative infarct size in a mouse model of myocardial ischemia/reperfusion injury indicating that the capacity for producing therapeutic exosomes was preserved. CONCLUSION: Our results demonstrated that MYC transformation is a practical strategy in ensuring an infinite supply of cells for the production of exosomes in the milligram range as either therapeutic agents or delivery vehicles. In addition, the increased proliferative rate by MYC transformation reduces the time for cell production and thereby reduces production costs. (hide) EV-METRIC 0% (median: 14% 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 HPLC Protein markers EV: Actin/ 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 Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Other Name other separation method HPLC Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ Actin ELISA Antibody details provided? No Detected EV-associated proteins Actin Characterization: Particle analysis DLS

	EV110087	1/1	Homo sapiens	Semen	(d)(U)C SEC	Carlsson L	2011	0%
Study summary Full title Association of cystatin C with prostasomes in human seminal plasma All authors Carlsson L, Ronquist G, Ronquist G, Eliasson R, Egberg N, Larsson A Journal Int J Androl Abstract It was recently elucidated that cystatin C, a protein targeted to the classical secretory pathway by (show more...)It was recently elucidated that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, can also be secreted in association with exosomes. Accordingly, we wanted to investigate whether there is a secretory link between cystatin C and prostasomes in human seminal plasma. Cystatin C concentrations in seminal plasma from 50 men including 6 vasectomized men were measured by turbidimetry on an Architect Ci8200. Some of the seminal plasma samples were also analysed utilizing an Epics Profile XL-MCL cytometer. We found high concentrations of cystatin C in seminal plasma. The 2.5-97.5 percentiles, performed by bootstrap estimation, were 25.8 [95% confidence interval (CI): 22.3-29.4] to 77.0 mg/L (95% CI: 71.9-82.1). Cystatin C is present in approximately 50 times higher concentration in seminal plasma compared with blood plasma. There was no clear difference as regards seminal plasma content of cystatin C between vasectomized men and the rest of the group. Immunoblot analysis with chicken anti-cystatin C antibody revealed a firm association of cystatin C with prostasomes. Flow cytometric analysis demonstrated that cystatin C was linked to prostasomes also meaning an at least partial prostasomal membrane surface localization. (hide) EV-METRIC 0% (median: 25% 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 Prostasomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C SEC Protein markers EV: non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Semen Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Particle analysis None

	EV110086	1/1	Shigella flexneri	Bacteria	(d)(U)C Filtration UF	Camacho AI	2011	0%
Study summary Full title Mucosal immunization with Shigella flexneri outer membrane vesicles induced protection in mice All authors Camacho AI, de Souza J, Sánchez-Gómez S, Pardo-Ros M, Irache JM, Gamazo C Journal Vaccine Abstract Vaccination appears to be the only rational prophylactic approach to control shigellosis. Unfortunat (show more...)Vaccination appears to be the only rational prophylactic approach to control shigellosis. Unfortunately, there is still no safe and efficacious vaccine available. We investigated the protection conferred by a new vaccine containing outer membrane vesicles (OMVs) from Shigella flexneri with an adjuvant based on nanoparticles in an experimental model of shigellosis in mice. OMVs were encapsulated in poly(anhydride) nanoparticles prepared by a solvent displacement method with the copolymer PMV/MA. OMVs loaded into NPs (NP-OMVs) were homogeneous and spherical in shape, with a size of 197nm (PdI=0.06). BALB/c mice (females, 9-week-old, 20±1g) were immunized by intradermal, nasal, ocular (20?g) or oral route (100?g) with free or encapsulated OMV. Thirty-five days after administration, mice were infected intranasally with a lethal dose of S. flexneri (1×10(7)CFU). The new vaccine was able to protect fully against infection when it was administered via mucosa. By intradermal route the NP-OMVs formulation increased the protection from 20%, obtained with free extract, to 100%. Interestingly, both OMVs and OMV-NP induced full protection when administered by the nasal and conjuntival route. A strong association between the ratio of IL-12p40/IL-10 and protection was found. Moreover, low levels of IFN-? correlate with protection. Under the experimental conditions used, the adjuvant did not induce any adverse effects. These results place OMVs among promising candidates to be used for vaccination against Shigellosis. (hide) EV-METRIC 0% (median: 14% of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bacteria Sample origin NAY Focus vesicles OMV Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Shigella flexneri Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: time(min) 120 Filtration steps 0.45µm > x > 0.22µm, Characterization: Particle analysis EM EM-type scanning EM Image type Wide-field Report size (nm) Not reported

	EV110085	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Batagov AO	2011	0%
Study summary Full title Identification of nucleotide patterns enriched in secreted RNAs as putative cis-acting elements targeting them to exosome nano-vesicles All authors Batagov AO, Kuznetsov VA, Kurochkin IV Journal BMC Genomics Abstract BACKGROUND: Exosomes are nanoscale membrane vesicles released by most cells. They are postulated to (show more...)BACKGROUND: Exosomes are nanoscale membrane vesicles released by most cells. They are postulated to be involved in cell-cell communication and genetic reprogramming of their target cells. In addition to proteins and lipids, they release RNA molecules many of which are not present in the donor cells implying a highly selective mode of their packaging into these vesicles. Sequence motifs targeting RNA to the vesicles are currently unknown. RESULTS: Ab initio approach was applied for computational identification of potential RNA secretory motifs in the primary sequences of exosome-enriched RNAs (eRNAs). Exhaustive motif analysis for the first time revealed unique sequence features of eRNAs. We discovered multiple linear motifs specifically enriched in secreted RNAs. Their potential function as cis-acting elements targeting RNAs to exosomes is proposed. The motifs co-localized in the same transcripts suggesting combinatorial organization of these secretory signals. We investigated associations of the discovered motifs with other RNA parameters. Secreted RNAs were found to have almost twice shorter half-life times on average, in comparison with cytoplasmic RNAs, and the occurrence of some eRNA-specific motifs significantly correlated with this eRNA feature. Also, we found that eRNAs are highly enriched in long noncoding RNAs. CONCLUSIONS: Secreted RNAs share specific sequence motifs that may potentially function as cis-acting elements targeting RNAs to exosomes. Discovery of these motifs will be useful for our understanding the roles of eRNAs in cell-cell communication and genetic reprogramming of the target cells. It will also facilitate nano-scale vesicle engineering and selective targeting of RNAs of interest to these vesicles for gene therapy purposes. (hide) EV-METRIC 0% (median: 14% 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: non-EV: 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Wash: volume per pellet (ml) 13 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis None

	EV110025	2/2	Homo sapiens	Blood plasma		Aung T	2011	0%
Study summary Full title Exosomal evasion of humoral immunotherapy in aggressive B-cell lymphoma modulated by ATP-binding cassette transporter A3 All authors Aung T, Chapuy B, Vogel D, Wenzel D, Oppermann M, Lahmann M, Weinhage T, Menck K, Hupfeld T, Koch R, Trümper L, Wulf GG Journal Proc Natl Acad Sci U S A Abstract Targeting the surface of malignant cells has evolved into a cornerstone in cancer therapy, paradigma (show more...)Targeting the surface of malignant cells has evolved into a cornerstone in cancer therapy, paradigmatically introduced by the success of humoral immunotherapy against CD20 in malignant lymphoma. However, tumor cell susceptibility to immunochemotherapy varies, with mostly a fatal outcome in cases of resistant disease. Here, we show that lymphoma exosomes shield target cells from antibody attack and that exosome biogenesis is modulated by the lysosome-related organelle-associated ATP-binding cassette (ABC) transporter A3 (ABCA3). B-cell lymphoma cells released exosomes that carried CD20, bound therapeutic anti-CD20 antibodies, consumed complement, and protected target cells from antibody attack. ABCA3, previously shown to mediate resistance to chemotherapy, was critical for the amounts of exosomes released, and both pharmacological blockade and the silencing of ABCA3 enhanced susceptibility of target cells to antibody-mediated lysis. Mechanisms of cancer cell resistance to drugs and antibodies are linked in an ABCA3-dependent pathway of exosome secretion. (hide) EV-METRIC 0% (median: 22% 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 Protein markers EV: CD63/ CD9 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method Other Name other separation method Characterization: Particle analysis None

	EV110084	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Atay S	2011	0%
Study summary Full title Human trophoblast-derived exosomal fibronectin induces pro-inflammatory IL-1beta production by macrophages All authors Atay S, Gercel-Taylor C, Taylor DD Journal Am J Reprod Immunol Abstract PROBLEM Our previous studies demonstrated that trophoblast-derived exosomes induced synthesis and re (show more...)PROBLEM Our previous studies demonstrated that trophoblast-derived exosomes induced synthesis and release of pro-inflammatory cytokines, including interleukin-1? (IL-1?) by macrophages. The objective of this study was to characterize the mechanism and receptors associated with this induction. METHOD OF STUDY Exosomes were isolated from Sw71 trophoblast-conditioned media by ultrafiltration and ultracentrifugation. Using macrophages isolated from normal donors, cytochalasin D was used to block exosome uptake. Induction of IL-1? mRNA was investigated by qRT-PCR, pro-IL-1? protein by western immunoblotting, and mature IL-1? release by ELISA. RGD peptides were used to block fibronectin binding by macrophage ?5?1 integrin. RESULTS Uptake of exosomes by macrophages was completely blocked by pre-treatment with cytochalasin D. Although induction of some cytokines (such as C4A and CCL11) requires uptake, induction of IL-1? occurred without exosome internalization. Cytochalasin D treatment did not inhibit exosome-mediated induction of IL-1? mRNA, production of the pro-protein, or release of mature IL-1?. Blocking of fibronectin binding using RGD peptides demonstrated the abrogation of exosome-mediated IL-1? production. CONCLUSION Although trophoblast-derived exosomes have been demonstrated to induce IL-1?, this is the first demonstration of IL-1? induction by exosome-associated fibronectin. Based on this pro-inflammatory role of exosome-associated fibronectin, it may represent an important general immunoregulatory mechanism. (hide) EV-METRIC 0% (median: 14% 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: 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis None

	EV110083	1/1	Homo sapiens	NAY	(d)(U)C UF	Atay S	2011	0%
Study summary Full title Trophoblast-derived exosomes mediate monocyte recruitment and differentiation All authors Atay S, Gercel-Taylor C, Suttles J, Mor G, Taylor DD Journal Am J Reprod Immunol Abstract INTRODUCTION: trophoblast cells have been demonstrated to regulate monocyte migration and differenti (show more...)INTRODUCTION: trophoblast cells have been demonstrated to regulate monocyte migration and differentiation, leading to pro-inflammatory profiles. Because trophoblast cells release exosomes with immunoregulatory properties, trophoblast-derived exosomes are proposed to 'educate' monocytes, creating a pro-inflammatory environment. METHOD OF STUDY: exosomes were isolated from conditioned media of Swan71 cells by ultrafiltration and ultracentrifugation. Exosome-induced migration was assessed using a two-chamber system. Cytokine profiles were defined using cytokine arrays, and mRNA levels of affected cytokines were examined by qRT-PCR and ELISA. RESULTS: within 20 min, 8-10% of monocytes took up labeled exosomes isolated from Swan71 cells. Trophoblast-derived exosomes increased monocyte migration in a dose-dependent manner and produced significant increases in production of interleukin (IL)-1?, IL-6, Serpin-E1, granulocyte colony-stimulating factor, granulocyte/monocyte colony-stimulating factor, and tumor necrosis factor-?. CONCLUSION: this study presents the initial demonstration that trophoblast-derived exosomes are capable of recruiting and 'educating' monocytes to produce pro-inflammatory cytokine/chemokine profiles in a cell-contact-independent manner. (hide) EV-METRIC 0% (median: 14% of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: non-EV: 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Particle analysis None

	EV110076	1/3	Homo sapiens	Blood plasma	(d)(U)C	Arroyo JD	2011	0%
Study summary Full title Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma All authors Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF, Tewari M Journal Proc Natl Acad Sci U S A Abstract MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being (show more...)MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation. (hide) EV-METRIC 0% (median: 22% 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 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 Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV110076	3/3	Homo sapiens	Serum		Arroyo JD	2011	0%
Study summary Full title Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma All authors Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF, Tewari M Journal Proc Natl Acad Sci U S A Abstract MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being (show more...)MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non-vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2-miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation. (hide) EV-METRIC 0% (median: 13% 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 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 Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method Other Name other separation method Characterization: Particle analysis None

	EV110082	1/1	Homo sapiens	NAY	(d)(U)C	Akao Y	2011	0%
Study summary Full title Microvesicle-mediated RNA molecule delivery system using monocytes/macrophages All authors Akao Y, Iio A, Itoh T, Noguchi S, Itoh Y, Ohtsuki Y, Naoe T Journal Mol Ther Abstract Microvesicles (MVs) and exosomes, which are shed from cells as a cell-to-cell communication tool, ar (show more...)Microvesicles (MVs) and exosomes, which are shed from cells as a cell-to-cell communication tool, are possible vehicles for navigating RNA molecules to body tissues. It is considered that intravenous injection of such MVs or exosomes from patients would not cause severe not-self and toxic reactions. Previously, we found that macrophages take up liposome-entrapped RNA molecules, some of which remain undegraded in the cells. Here, we demonstrate that transfected RNA molecules in human monocytic leukemia THP-1 cells were shed from THP-1 macrophages as contents in MVs during incubation in serum-free medium, which shedding was shown by biochemical analyses such as quantitative reverse transcription (qRT)-PCR, expression of TSG101 (a membrane-associated exosomal protein), and immunoelectron microscopic study. More chemically modified RNA molecules (miR-143BPs) entrapped by MVs (MV-miR-143BPs) were secreted from THP-1 macrophages after miR-143BP transfection compared with the amount after transfection with nonmodified miR-143 transfection. Furthermore, we show that the THP-1 macrophages, which were transfected with the miR-143BP ex vivo, secreted MV-miR-143BPs in xenografted nude mice after intravenous injection, because miR-143 levels were significantly increased in the serum, tumor, and kidney of the host animals. These data suggest that some of the transfected miR-143BPs were secreted from THP-1 macrophages as MV-RNAs both in vitro and in vivo. (hide) EV-METRIC 0% (median: 14% 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 Protein markers EV: TSG101/ Beta-actin 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 Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 180 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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