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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140207	1/1	Homo sapiens	NAY	(d)(U)C Filtration IAF	Pook M	2014	33%
Study summary Full title Platelets store laminins 411/421 and 511/521 in compartments distinct from alpha- or dense granules and secrete these proteins via microvesicles All authors Pook M, Tamming L, Padari K, Tiido T, Maimets T, Patarroyo M, Juronen E, Jaks V, Ingerpuu S Journal J Thromb Haemost Abstract BACKGROUND: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive (show more...)BACKGROUND: Blood platelets secrete upon activation of laminins 411/421 and 511/521, large adhesive proteins mainly found in the basement membranes of blood vessels and other tissues. At present, the subcellular localization and secretion mechanisms of platelet laminins are largely unknown. OBJECTIVES: Our aim was to compare the subcellular localization of laminins 411/421 and 511/521 and specific granule markers in platelets. We also elucidated the role of microvesicles and exosomes in laminin release in platelet activation. METHODS: We studied laminin and granule marker protein localization in platelets by using immunofluorescence confocal microscopy and immunoelectron microscopy. Microvesicles and exosomes were separated from material released from platelets on activation by thrombin. The expression of laminins in microvesicles and exosomes was studied by using SDS-PAGE and Western blotting as well as by flow cytometric analysis. The exosomes were immunoprecipitated with magnetic microbeads coated with anti-CD63 antibodies. RESULTS AND CONCLUSIONS: We demonstrate that laminins 411/421 and 511/521 are present in compartments of platelets that do not express ?-granule, dense granule, or lysosome marker proteins. Moreover, laminins secreted by activated platelets are mostly found in microvesicles shed from the plasma membrane, while their presence in simultaneously released exosomes is minimum. (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 Filtration IAF Adj. k-factor 255.8 (pelleting) Protein markers EV: CD63/ CD41/ CD62P/ CD61 non-EV: Proteomics no Show all info Study aim Other/Laminin secretion Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Filtration steps 0.2µm > x > 0.1µm Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD41/ CD61/ CD62P ELISA Antibody details provided? No Detected EV-associated proteins CD41/ CD61/ CD62P

	EV140035	2/2	Homo sapiens	Serum	(d)(U)C Filtration	Melo SA	2014	33%
Study summary Full title Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis All authors Melo SA, Sugimoto H, O'Connell JT, Kato N, Villanueva A, Vidal A, Qiu L, Vitkin E, Perelman LT, Melo CA, Lucci A, Ivan C, Calin GA, Kalluri R Journal Cancer Cell Abstract Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that (show more...)Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. (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 Filtration Adj. k-factor 276.6 (pelleting) / 276.6 (washing) Protein markers EV: Flotilin1 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens 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) 120 Pelleting: rotor type SW40 Pelleting: adjusted k-factor 276.6 Wash: Rotor Type SW40 Wash: adjusted k-factor 276.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Flotilin1 Characterization: Particle analysis NTA EM EM-type transmission EM/ atomic force EM Image type Close-up

	EV140052	1/2	Homo sapiens	NAY	(d)(U)C DC Filtration	Lundholm M	2014	33%
Study summary Full title Prostate tumor-derived exosomes down-regulate NKG2D expression on natural killer cells and CD8+ T cells: mechanism of immune evasion All authors Lundholm M, Schröder M, Nagaeva O, Baranov V, Widmark A, Mincheva-Nilsson L, Wikström P Journal PLoS One Abstract Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have e (show more...)Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8+ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8+ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8+ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape. (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 Protein markers EV: TSG101/ PSMA/ 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ PSMA Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins PSMA Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140033	1/1	Homo sapiens	NAY	(d)(U)C Filtration UF	Liu C	2014	33%
Study summary Full title Unconventional secretion of synuclein-gamma promotes tumor cell invasion All authors Liu C, Qu L, Lian S, Tian Z, Zhao C, Meng L, Shou C Journal FEBS J Abstract Synuclein-? (SNCG) is a chaperone protein and exists mainly in the cytoplasm. SNCG confers chemoresi (show more...)Synuclein-? (SNCG) is a chaperone protein and exists mainly in the cytoplasm. SNCG confers chemoresistance, and is a potential unfavorable biomarker for multiple types of cancer. Our previous work demonstrated that SNCG could be detected in the serum of cancer patients and the medium of cultured cancer cells, but the mechanism of SNCG secretion and its biological roles are unknown. Here, we showed that SNCG levels in the culture medium were positively correlated with cancer cell densities and the concentrations of fetal bovine serum added. SNCG secretion was unaffected by brefeldin A, an inhibitor of the classic protein transport pathway, but was antagonized by exosome inhibitor, lysosome inhibitor, ABC transporter inhibitor, and phosphatidylinositide 3-kinase inhibitor, and knockdown of Rab27a. Ultracentrifugation fractionation revealed that intracellular SNCG was present as both free and vesicle-associated forms, but that the extracellular SNCG was mainly free. The results of reciprocal coimmunoprecipitation experiments showed an interaction between SNCG and flotillin-2, a marker of exosomes and lipid rafts. Moreover, we demonstrated that SNCG, both secreted from tumor cells and exogenously added, markedly promoted cancer cell migration and invasion, but had no effect on noncancerous cells. These findings suggest that SNCG is actively secreted by cancer cells via an unconventional secretion pathway and contributes to aggressive phenotypes of cancer cells. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Protein markers EV: HSP90/ GAPDH/ Alix/ Annexin2/ LAMP1/ Flotillin2/ HSP70 non-EV: Cell organelle protein Proteomics no Show all info Study aim Other/Synuclein-gamma secretion Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP90/ HSP70/ "Flotillin2/ LAMP1/ Annexin2/ GAPDH" Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins "Flotillin2/ LAMP1/ Annexin2/ GAPDH" Characterization: Particle analysis None

	EV140184	1/1	Homo sapiens	NAY	(d)(U)C DG UF	Kuruppu S	2014	33%
Study summary Full title Nitric oxide inhibits the production of soluble endothelin converting enzyme-1 All authors Kuruppu S, Rajapakse NW, Dunstan RA, Smith AI Journal Mol Cell Biochem Abstract This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 (show more...)This study examined the effect of nitric oxide on the production of soluble ECE-1. Activity of ECE-1 in media was measured using a quenched fluorescent substrate assay, and expressed as a percentage of control. Endothelial cells were incubated with the nitric oxide donor Diethylenetriamine NONOate (DETA; 250-800 µM), NOS substrate L-Arg (200-1,000 µM), a L-Arg transport inhibitor (L-Lys; 10 µM) and NOS inhibitors (L-Gln and N5-[imino(nitroamino)methyl]-L-ornithine, methyl ester, monohydrochloride (L-NAME); 10-100 µM). The effect of L-Arg (1,000 µM) was also tested in the presence of L-Lys (10 µM), L-Gln (100 µM) and L-NAME (10-100 µM). Ultracentrifugation (100,000×g, 4 °C, 1 h) completely removed ECE-1 activity from the supernatant. In addition, fractionation of concentrated media on a sucrose density gradient indicated that ECE-1 activity was localised to the mid portion of the gradient, thus suggesting the possible role of exosomes in ECE-1 release. Production of soluble ECE-1 by Ea.hy926 cells was inhibited significantly (P < 0.05, unpaired t test, n = 4) in the presence of DETA (75.31 ± 3.59; 800 µM) and L-Arg (60.97 ± 9.22; 1,000 µM). L-Arg-mediated reduction in the release of soluble ECE-1 was blocked by the inhibition of NOS using L-NAME (100 µM; 99.19 ± 0.58) and L-Gln (100 µM; 104.41 ± 0.65). In addition, the presence of L-Lys (10 µM) significantly blocked the L-Arg (1,000 µM)-induced reduction in soluble ECE-1 levels (122.38 ± 13.16). These treatments had no effect on the expression of ECE-1 on the cell surface. Our data provide evidence that NO can inhibit the production of soluble ECE-1 by endothelial cells. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG UF Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.38-1.4 Show all info Study aim Other/ECE-1 release 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 Density gradient Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Characterization: Particle analysis None

	EV140181	1/1	Homo sapiens	Blood plasma	(d)(U)C DG	Konadu KA	2014	33%
Study summary Full title Cytokines Associate with Exosomes in the Plasma of HIV-1+ Individuals All authors Konadu KA, Chu J, Huang MB, Amancha PK, Armstrong W, Powell MD, Villinger F, Bond VC Journal J Infect Dis Abstract Human immunodeficiency virus (HIV)-infected and viremic individuals exhibit elevated levels of plasm (show more...)Human immunodeficiency virus (HIV)-infected and viremic individuals exhibit elevated levels of plasma cytokines. Here we show that most cytokines are not in free form but appear associated with exosomes that are distinct from virions. Purified exosomes were analyzed to determine the levels of 21 cytokines and chemokines and compared with exosome-depleted plasma. Most cytokines were markedly enriched in exosomes from HIV-positive individuals relative to negative controls and to plasma. Moreover, exposure of naive peripheral blood mononuclear cells to exosomes purified from HIV-positive patients induced CD38 expression on naive and central memory CD4(+) and CD8(+) T cells, probably contributing to inflammation and viral propagation via bystander cell activation. (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 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: CD45/ AChE/ CD63/ CD9 non-EV: p24 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Density gradient Speed (g) 400000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD45/ AChE Detected contaminants p24 ELISA Antibody details provided? No Detected EV-associated proteins CD45/ AChE Characterization: Particle analysis EM EM-type immune EM EM protein CD63 Image type Close-up

	EV140028	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Jaworski E	2014	33%
Study summary Full title Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein All authors Jaworski E, Narayanan A, Van Duyne R, Shabbeer-Meyering S, Iordanskiy S, Saifuddin M, Das R, Afonso PV, Sampey GC, Chung M, Popratiloff A, Shrestha B, Sehgal M, Jain P, Vertes A, Mahieux R, Kashanchi F Journal J Biol Chem Abstract Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV- (show more...)Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-?B in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: Alix/ HSP70/ Beta-actin/ CD63 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Wash: volume per pellet (ml) 22 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ HSP70/ Beta-actin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140273	2/2	Homo sapiens	NAY	Filtration Tangential flow filter and track etch filter UF	Heinemann ML	2014	33%
Study summary Full title Benchtop isolation and characterization of functional exosomes by sequential filtration All authors Heinemann ML, Ilmer M, Silva LP, Hawke DH, Recio A, Vorontsova MA, Alt E, Vykoukal J Journal J Chromatogr A Abstract Early and minimally invasive detection of malignant events or other pathologies is of utmost importa (show more...)Early and minimally invasive detection of malignant events or other pathologies is of utmost importance in the pursuit of improved patient care and outcomes. Recent evidence indicates that exosomes and extracellular vesicles in serum and body fluids can contain nucleic acid, protein, and other biomarkers. Accordingly, there is great interest in applying these clinically as prognostic, predictive, pharmacodynamic, and early detection indicators. Nevertheless, existing exosome isolation methods can be time-consuming, require specialized equipment, and/or present other inefficiencies regarding purity, reproducibility and assay cost. We have developed a straightforward, three-step protocol for exosome isolation of cell culture supernatants or large volumes of biofluid based on sequential steps of dead-end pre-filtration, tangential flow filtration (TFF), and low-pressure track-etched membrane filtration that we introduce here. Our approach yields exosome preparations of high purity and defined size distribution and facilitates depletion of free protein and other low-molecular-weight species, extracellular vesicles larger than 100nm, and cell debris. Samples of exosomes prepared using the approach were verified morphologically by nanoparticle tracking analysis and electron microscopy, and mass spectrometry analyses confirmed the presence of previously reported exosome-associated proteins. In addition to being easy-to-implement, sequential filtration yields exosomes of high purity and, importantly, functional integrity as a result of the relatively low-magnitude manipulation forces employed during isolation. This answers an unmet need for preparation of minimally manipulated exosomes for investigations into exosome function and basic biology. Further, the strategy is amenable to translation for clinical exosome isolations because of its speed, automatability, scalability, and specificity for isolating exosomes from complex biological samples. (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 Filtration Tangential flow filter and track etch filter UF Protein markers EV: non-EV: Proteomics yes Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method Filtration steps 0.2µm > x > 0.1µm Other Name other separation method Tangential flow filter and track etch filter Characterization: Protein analysis Characterization: Particle analysis DLS NTA EM EM-type immune EM EM protein CD63 Image type Close-up

	EV140117	3/8	Homo sapiens	Blood plasma	IAF Microfluidics	He M	2014	33%
Study summary Full title Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology All authors He M, Crow J, Roth M, Zeng Y, Godwin AK Journal Lab Chip Abstract Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy (show more...)Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy is difficult, costly, and sometimes not even an option. Tumor-derived exosomes have attracted increasing interest in non-invasive cancer diagnosis and monitoring of treatment response. However, the biology and clinical value of exosomes remains largely unknown due in part to current technical challenges in rapid isolation, molecular classification and comprehensive analysis of exosomes. Here we developed a new microfluidic approach to streamline and expedite the exosome analysis pipeline by integrating specific immunoisolation and targeted protein analysis of circulating exosomes. Compared to the conventional methods, our approach enables selective subpopulation isolation and quantitative detection of surface and intravesicular biomarkers directly from a minimally invasive amount of plasma samples (30 ?L) within ~100 min with markedly improved detection sensitivity. Using this device, we demonstrated phenotyping of exosome subpopulations by targeting a panel of common exosomal and tumor-specific markers and multiparameter analyses of intravesicular biomarkers in the selected subpopulation. We were able to assess the total expression and phosphorylation levels of IGF-1R in non-small-cell lung cancer patients by probing plasma exosomes as a non-invasive alternative to conventional tissue biopsy. We foresee that the microfluidic exosome analysis platform will form the basis for critically needed infrastructures for advancing the biology and clinical utilization of exosomes. (hide) EV-METRIC 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 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 IAF Microfluidics Protein markers EV: non-EV: Proteomics no TEM measurements 50-100 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method Immunoaffinity capture Selected surface protein(s) CD81 Other Name other separation method Microfluidics Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV140117	4/8	Homo sapiens	Blood plasma	IAF Microfluidics	He M	2014	33%
Study summary Full title Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology All authors He M, Crow J, Roth M, Zeng Y, Godwin AK Journal Lab Chip Abstract Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy (show more...)Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy is difficult, costly, and sometimes not even an option. Tumor-derived exosomes have attracted increasing interest in non-invasive cancer diagnosis and monitoring of treatment response. However, the biology and clinical value of exosomes remains largely unknown due in part to current technical challenges in rapid isolation, molecular classification and comprehensive analysis of exosomes. Here we developed a new microfluidic approach to streamline and expedite the exosome analysis pipeline by integrating specific immunoisolation and targeted protein analysis of circulating exosomes. Compared to the conventional methods, our approach enables selective subpopulation isolation and quantitative detection of surface and intravesicular biomarkers directly from a minimally invasive amount of plasma samples (30 ?L) within ~100 min with markedly improved detection sensitivity. Using this device, we demonstrated phenotyping of exosome subpopulations by targeting a panel of common exosomal and tumor-specific markers and multiparameter analyses of intravesicular biomarkers in the selected subpopulation. We were able to assess the total expression and phosphorylation levels of IGF-1R in non-small-cell lung cancer patients by probing plasma exosomes as a non-invasive alternative to conventional tissue biopsy. We foresee that the microfluidic exosome analysis platform will form the basis for critically needed infrastructures for advancing the biology and clinical utilization of exosomes. (hide) EV-METRIC 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 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 IAF Microfluidics Protein markers EV: non-EV: Proteomics no TEM measurements 25-100 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method Immunoaffinity capture Selected surface protein(s) CD9 Other Name other separation method Microfluidics Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field Report size (nm) 25-100

	EV140117	8/8	Homo sapiens	Blood plasma	IAF Microfluidics	He M	2014	33%
Study summary Full title Integrated immunoisolation and protein analysis of circulating exosomes using microfluidic technology All authors He M, Crow J, Roth M, Zeng Y, Godwin AK Journal Lab Chip Abstract Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy (show more...)Developing blood-based tests is appealing for non-invasive disease diagnosis, especially when biopsy is difficult, costly, and sometimes not even an option. Tumor-derived exosomes have attracted increasing interest in non-invasive cancer diagnosis and monitoring of treatment response. However, the biology and clinical value of exosomes remains largely unknown due in part to current technical challenges in rapid isolation, molecular classification and comprehensive analysis of exosomes. Here we developed a new microfluidic approach to streamline and expedite the exosome analysis pipeline by integrating specific immunoisolation and targeted protein analysis of circulating exosomes. Compared to the conventional methods, our approach enables selective subpopulation isolation and quantitative detection of surface and intravesicular biomarkers directly from a minimally invasive amount of plasma samples (30 ?L) within ~100 min with markedly improved detection sensitivity. Using this device, we demonstrated phenotyping of exosome subpopulations by targeting a panel of common exosomal and tumor-specific markers and multiparameter analyses of intravesicular biomarkers in the selected subpopulation. We were able to assess the total expression and phosphorylation levels of IGF-1R in non-small-cell lung cancer patients by probing plasma exosomes as a non-invasive alternative to conventional tissue biopsy. We foresee that the microfluidic exosome analysis platform will form the basis for critically needed infrastructures for advancing the biology and clinical utilization of exosomes. (hide) EV-METRIC 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 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 IAF Microfluidics Protein markers EV: non-EV: Proteomics no TEM measurements 50-100 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Separation Method Immunoaffinity capture Selected surface protein(s) Î±-IGF-1R Other Name other separation method Microfluidics Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV140078	1/1	Mus musculus	NAY	(d)(U)C Filtration	Guo BB	2014	33%
Study summary Full title The neutral sphingomyelinase pathway regulates packaging of the prion protein into exosomes All authors Guo BB, Bellingham SA, Hill AF Journal J Biol Chem Abstract Prion diseases are a group of transmissible, fatal neurodegenerative disorders associated with the m (show more...)Prion diseases are a group of transmissible, fatal neurodegenerative disorders associated with the misfolding of the host-encoded prion protein, PrP(C), into a disease-associated form, PrP(Sc). The transmissible prion agent is principally formed of PrP(Sc) itself and is associated with extracellular vesicles known as exosomes. Exosomes are released from cells both in vitro and in vivo, and have been proposed as a mechanism by which prions spread intercellularly. The biogenesis of exosomes occurs within the endosomal system, through formation of intraluminal vesicles (ILVs), which are subsequently released from cells as exosomes. ILV formation is known to be regulated by the endosomal sorting complexes required for transport (ESCRT) machinery, although an alternative neutral sphingomyelinase (nSMase) pathway has been suggested to also regulate this process. Here, we investigate a role for the nSMase pathway in exosome biogenesis and packaging of PrP into these vesicles. Inhibition of the nSMase pathway using GW4869 revealed a role for the nSMase pathway in both exosome formation and PrP packaging. In agreement, targeted knockdown of nSMase1 and nSMase2 in mouse neurons using lentivirus-mediated RNAi also decreases exosome release, demonstrating the nSMase pathway regulates the biogenesis and release of exosomes. We also demonstrate that PrP(C) packaging is dependent on nSMase2, whereas the packaging of disease-associated PrP(Sc) into exosomes occurs independently of nSMase2. These findings provide further insight into prion transmission and identify a pathway which directly assists exosome-mediated transmission of prions. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ AChE/ Flotilin1 non-EV: NUCLEOPORIN/ Cell organelle protein/ Bcl2 Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ TSG101/ AChE Detected contaminants Cell organelle protein/ "Bcl2/ NUCLEOPORIN" ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV140075	1/1	Mus musculus	NAY	(d)(U)C DG	Glebov K	2014	33%
Study summary Full title Serotonin stimulates secretion of exosomes from microglia cells All authors Glebov K, Löchner M, Jabs R, Lau T, Merkel O, Schloss P, Steinhäuser C, Walter J Journal Glia Abstract Microglia are resident immune cells in the brain and exert important functions in the regulation of (show more...)Microglia are resident immune cells in the brain and exert important functions in the regulation of inflammatory processes during infection or cellular damage. Upon activation, microglia undergo complex morphological and functional transitions, including increased motility, phagocytosis and cytokine secretion. Recent findings indicate that exosomes, small vesicles that derive from fusion of multivesicular bodies with the plasma membrane, are involved in secretion of certain cytokines. The presence of specific receptors on the surface of microglia suggests communication with neurons by neurotransmitters. Here, we demonstrate expression of serotonin receptors, including 5-HT2a,b and 5-HT4 in microglial cells and their functional involvement in the modulation of exosome release by serotonin. Our data demonstrate the involvement of cAMP and Ca(2+) dependent signaling pathways in the regulation of exosome secretion. Co-culture of microglia with embryonic stem cell-derived serotonergic neurons further demonstrated functional signaling between neurons and microglia. Together, these data provide evidence for neurotransmitter-dependent signaling pathways in microglial cells that regulate exosome release. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ Beta-actin/ Flotilin1 non-EV: 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 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ Flotilin1/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins Beta-actin Characterization: Particle analysis None

	EV140073	1/1	Homo sapiens	Blood plasma	(d)(U)C ExoQuick Filtration	Ge Q	2014	33%
Study summary Full title miRNA in plasma exosome is stable under different storage conditions All authors Ge Q, Zhou Y, Lu J, Bai Y, Xie X, Lu Z Journal Molecules Abstract Exosomes are small membrane-bound vesicles secreted by most cell types. Exosomes contain various fun (show more...)Exosomes are small membrane-bound vesicles secreted by most cell types. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs) that could be used for diagnostic and therapeutic purposes. How we should store the samples before RNA isolation and whether those long term stored samples could be used for circulating RNA investigation because of RNase is unknown. The aim of the study was to determine the stability of circulating miRNA in exosomes and plasma. Exosomes were isolated from plasma samples by using ExoQuick Precipitation methods. RNA was extracted from exosomes and the corresponding plasma samples with a Qiagen miRNeasy Mini kit. The concentration of RNA was measured by a Qubit® RNA HS Assay Kit, and quantitative PCR was used for individual miRNA expression level detection. Results showed that exosomal miRNA showed extra stability under different storage conditions and no significant influence on plasma miRNA, except for short term storage at 4 °C. It is thus indicated that exosome miRNAs can be good biomarkers based on their stability under various storage conditions. (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 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: 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 10,000 g and 50,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

	EV140072	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Franquesa M	2014	33%
Study summary Full title Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells All authors Franquesa M, Hoogduijn MJ, Ripoll E, Luk F, Salih M, Betjes MG, Torras J, Baan CC, Grinyó JM, Merino AM Journal Front Immunol Abstract The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the th (show more...)The research field on extracellular vesicles (EV) has rapidly expanded in recent years due to the therapeutic potential of EV. Adipose tissue human mesenchymal stem cells (ASC) may be a suitable source for therapeutic EV. A major limitation in the field is the lack of standardization of the challenging techniques to isolate and characterize EV. The aim of our study was to incorporate new controls for the detection and quantification of EV derived from ASC and to analyze the applicability and limitations of the available techniques. ASC were cultured in medium supplemented with 5% of vesicles-free fetal bovine serum. The EV were isolated from conditioned medium by differential centrifugation with size filtration (0.2 ?m). As a control, non-conditioned culture medium was used (control medium). To detect EV, electron microscopy, conventional flow cytometry, and western blot were used. The quantification of the EV was by total protein quantification, ExoELISA immunoassay, and Nanosight. Cytokines and growth factors in the EV samples were measured by multiplex bead array kit. The EV were detected by electron microscope. Total protein measurement was not useful to quantify EV as the control medium showed similar protein contents as the EV samples. The ExoELISA kits had technical troubles and it was not possible to quantify the concentration of exosomes in the samples. The use of Nanosight enabled quantification and size determination of the EV. It is, however, not possible to distinguish protein aggregates from EV with this method. The technologies for quantification and characterization of the EV need to be improved. In addition, we detected protein contaminants in the EV samples, which make it difficult to determine the real effect of EV in experimental models. It will be crucial in the future to optimize design novel methods for purification and characterization of EV. (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 Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: CD81/ CD63/ CD9 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: volume per pellet (ml) 39 Wash: Rotor Type 70Ti Wash: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140071	1/1	Mus musculus	NAY	(d)(U)C Filtration	Forterre A	2014	33%
Study summary Full title Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation All authors Forterre A, Jalabert A, Chikh K, Pesenti S, Euthine V, Granjon A, Errazuriz E, Lefai E, Vidal H, Rome S Journal Cell Cycle Abstract It has recently been established that exosomes can mediate intercellular cross-talk under normal and (show more...)It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as endocrine signals during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube-exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis. (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 Adj. k-factor 157.1 (pelleting) Protein markers EV: Alix/ CD81 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 502Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 25 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD81 Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein CD63 Image type Close-up, Wide-field

	EV140156	1/1	Homo sapiens	NAY	(d)(U)C DG	Feng Z	2014	33%
Study summary Full title Human pDCs preferentially sense enveloped hepatitis A virions All authors Feng Z, Li Y, McKnight KL, Hensley L, Lanford RE, Walker CM, Lemon SM Journal J Clin Invest Abstract Unlike other picornaviruses, hepatitis A virus (HAV) is cloaked in host membranes when released from (show more...)Unlike other picornaviruses, hepatitis A virus (HAV) is cloaked in host membranes when released from cells, providing protection from neutralizing antibodies and facilitating spread in the liver. Acute HAV infection is typified by minimal type I IFN responses; therefore, we questioned whether plasmacytoid dendritic cells (pDCs), which produce IFN when activated, are capable of sensing enveloped virions (eHAV). Although concentrated nonenveloped virus failed to activate freshly isolated human pDCs, these cells produced substantial amounts of IFN-? via TLR7 signaling when cocultured with infected cells. pDCs required either close contact with infected cells or exposure to concentrated culture supernatants for IFN-? production. In isopycnic and rate-zonal gradients, pDC-activating material cosedimented with eHAV but not membrane-bound acetylcholinesterase, suggesting that eHAV, and not viral RNA exosomes, is responsible for IFN-? induction. pDC activation did not require virus replication and was associated with efficient eHAV uptake, which was facilitated by phosphatidylserine receptors on pDCs. In chimpanzees, pDCs were transiently recruited to the liver early in infection, during or shortly before maximal intrahepatic IFN-stimulated gene expression, but disappeared prior to inflammation onset. Our data reveal that, while membrane envelopment protects HAV against neutralizing antibody, it also facilitates an early but limited detection of HAV infection by pDCs. (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 DG Protein markers EV: Alix/ AChE/ Flotilin1 non-EV: Proteomics no EV density (g/ml) 1.120 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) 60 Density gradient Lowest density fraction 8 Highest density fraction 40 Orientation Top-down Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ Flotilin1/ AChE ELISA Antibody details provided? No Detected EV-associated proteins AChE Characterization: Particle analysis None

	EV140154	1/1	Rattus norvegicus/rattus	NAY	(d)(U)C	Escudero CA	2014	33%
Study summary Full title The p75 neurotrophin receptor evades the endolysosomal route in neuronal cells, favouring multivesicular bodies specialised for exosomal release All authors Escudero CA, Lazo OM, Galleguillos C, Parraguez JI, Lopez-Verrilli MA, Cabeza C, Leon L, Saeed U, Retamal C, Gonzalez A, Marzolo MP, Carter BD, Court FA, Bronfman FC Journal J Cell Sci Abstract The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that r (show more...)The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and post-endocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 141.1 (pelleting) Protein markers EV: CD63/ Na/K ATPase/ Rab5/ B-COP non-EV: Cell organelle protein 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 Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type P55ST2 Pelleting: adjusted k-factor 141.1 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ "Na/K ATPase/ B-COP/ Rab5" Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins "Na/K ATPase/ B-COP/ Rab5" Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein p75 Image type Close-up

	EV140153	2/2	Homo sapiens	Blood plasma	(d)(U)C Filtration	Eldh M	2014	33%
Study summary Full title MicroRNA in exosomes isolated directly from the liver circulation in patients with metastatic uveal melanoma All authors Eldh M, Olofsson Bagge R, Lässer C, Svanvik J, Sjöstrand M, Mattsson J, Lindnér P, Choi DS, Gho YS, Lötvall J Journal BMC Cancer Abstract BACKGROUND: Uveal melanoma is a tumour arising from melanocytes of the eye, and 30 per cent of these (show more...)BACKGROUND: Uveal melanoma is a tumour arising from melanocytes of the eye, and 30 per cent of these patients develop liver metastases. Exosomes are small RNA containing nano-vesicles released by most cells, including malignant melanoma cells. This clinical translational study included patients undergoing isolated hepatic perfusion (IHP) for metastatic uveal melanoma, from whom exosomes were isolated directly from liver perfusates. The objective was to determine whether exosomes are present in the liver circulation, and to ascertain whether these may originate from melanoma cells. METHODS: Exosomes were isolated from the liver perfusate of twelve patients with liver metastases from uveal melanoma undergoing IHP. Exosomes were visualised by electron microscopy, and characterised by flow cytometry, Western blot and real-time PCR. Furthermore, the concentration of peripheral blood exosomes were measured and compared to healthy controls. RESULTS: The liver perfusate contained Melan-A positive and RNA containing exosomes, with similar miRNA profiles among patients, but dissimilar miRNA compared to exosomes isolated from tumor cell cultures. Patients with metastatic uveal melanoma had a higher concentration of exosomes in their peripheral venous blood compared to healthy controls. CONCLUSIONS: Melanoma exosomes are released into the liver circulation in metastatic uveal melanoma, and is associated with higher concentrations of exosomes in the systemic circulation. The exosomes isolated directly from liver circulation contain miRNA clusters that are different from exosomes from other cellular sources. (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 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 130.7 (pelleting) Protein markers EV: CD81/ Melan-A/ CD63/ CD9 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 Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 130.7 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81/ CD9/ Melan-A ELISA Antibody details provided? No Detected EV-associated proteins Melan-A 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 CD63 Image type Close-up

	EV140118	3/3	Sus scrofa	NAY	(d)(U)C Filtration	Comelli L	2014	33%
Study summary Full title Characterization of secreted vesicles from vascular smooth muscle cells All authors Comelli L, Rocchiccioli S, Smirni S, Salvetti A, Signore G, Citti L, Trivella MG, Cecchettini A Journal Mol Biosyst Abstract The artery medial layer is mainly composed of vascular smooth muscle cells (VSMCs). These cells cont (show more...)The artery medial layer is mainly composed of vascular smooth muscle cells (VSMCs). These cells contribute to the formation of neointima and atherosclerotic plaques by switching from the quiescent-contractile to migratory-activated state. Apoptotic blebs, microvesicles and exosomes are secreted vesicles, with differences in composition and size, involved in cellular communication at multiple levels. In this article, an untargeted, proteomics approach was exploited to characterise VSMC released vesicles and a preliminary protein profile for microvesicles and exosomes of different cell phenotypes was obtained. Enriched samples of vesicles from serum-free and serum-activated VSMCs were analysed by a LC-MS/MS strategy leading to the identification of 349 proteins. In microvesicles, the most abundant classes of identified proteins were cytoplasmic or organelle associated, house keeping and metabolic factors. Otherwise, exosomes from different phenotypes revealed a sharper peculiarity thus, as suggested by the high percentage of ECM and ECM related proteins and cell adhesion molecules, they seem to play an important role in outward or cell-to-cell signalling. Comparison between exosomes or microvesicles from quiescent and activated VSMCs evidenced 29 differentially expressed proteins. Among these, in microvesicles there are several proteins that are involved in vesicle trafficking while in exosomes focal adhesion and ECM related factors are the most interesting. These data, although preliminary, are promising for a possible identification of potential circulating markers of a cell state. (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 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: CD81/ HSP70/ TSG101/ Flotilin1 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Omics Sample Species Sus scrofa 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 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Flotilin1/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140066	1/1	Homo sapiens	"Cerebrospinal fluid"	(d)(U)C DC UF	Chiasserini D	2014	33%
Study summary Full title Proteomic analysis of cerebrospinal fluid extracellular vesicles: a comprehensive dataset All authors Chiasserini D, van Weering JR, Piersma SR, Pham TV, Malekzadeh A, Teunissen CE, de Wit H, Jiménez CR Journal J Proteomics Abstract Extracellular vesicles (EVs) are present in human cerebrospinal fluid (CSF), yet little is known abo (show more...)Extracellular vesicles (EVs) are present in human cerebrospinal fluid (CSF), yet little is known about their protein composition. The aim of this study is to provide a comprehensive analysis of the proteome of CSF EVs by electron microscopy and high resolution tandem mass spectrometry (MS/MS) in conjunction with bioinformatics. We report an extensive catalog of 1315 proteins identified in EVs isolated from two different CSF pools by ultracentrifugation, including 230 novel EV proteins. Out of 1315 proteins, 760 were identified in both CSF pools and about 30% of those were also quantitatively enriched in the EV fraction versus the soluble CSF fraction. The proteome of CSF EVs was enriched in exosomal markers such as alix and syntenin-1, heat shock proteins and tetraspanins and contained a high proportion of brain-derived proteins (n=373). Interestingly, several known biomarkers for neurodegenerative diseases such as the amyloid precursor protein, the prion protein and DJ-1 were identified in the EV fractions. Our dataset represents the first comprehensive inventory of the EV proteome in CSF, underscoring the biomarker potential of this organelle. Further comparative studies on CSF EVs isolated from patients diagnosed with neurological disorders are warranted. Data are available via ProteomeXchange with identifier PXD000608. Biological significance In this study we analyzed the protein composition of extracellular vesicles isolated from pooled samples of human cerebrospinal fluid (CSF). CSF is a colorless fluid surrounding the brain and the spinal cord, important for the physiology of the central nervous system, ensuing mechanical protection, regulation of brain blood flow and elimination of byproducts of the brain. Since brain (patho)physiology is reflected in CSF, this biological fluid represents an ideal source of soluble and vesicle-based biomarkers for neurological diseases. Here we confirm the presence of exosome-like extracellular vesicles in CSF, underscoring a potential role in the physiology of the brain. These extracellular vesicles provide a rich source of candidate biomarkers, representing a brain fluid biopsy. Most interestingly, the involvement of extracellular vesicles in transferring toxic proteins such as ?-synuclein and ?-amyloid has been postulated as one of the mechanisms involved in the spreading of neurodegeneration to different brain areas. In line with this, we show that human CSF extracellular vesicles contain prionogenic proteins such as the amyloid precursor protein and the prion protein. Delineating the protein composition of extracellular vesicles in CSF is a first and crucial step to comprehend their origin and their function in the central nervous system and to establish their biomarker potential. (hide) EV-METRIC 33% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type "Cerebrospinal fluid" 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 UF Adj. k-factor 276.6 (pelleting) / 276.6 (washing) Protein markers EV: Alix/ Flotilin1/ CD63/ Syntenin non-EV: Albumin Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type "Cerebrospinal fluid" 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 SW40 Pelleting: adjusted k-factor 276.6 Wash: Rotor Type SW40 Wash: adjusted k-factor 276.6 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ Flotilin1/ Syntenin Detected contaminants Albumin Characterization: Particle analysis EM EM-type immune EM EM protein CD63 Image type Close-up

	EV140065	1/2	Homo sapiens	Serum	(d)(U)C	Cheng L	2014	33%
Study summary Full title Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood All authors Cheng L, Sharples RA, Scicluna BJ, Hill AF Journal J Extracell Vesicles Abstract INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed (show more...)INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases. BACKGROUND: Next-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays. METHODS: Here, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA. CONCLUSION: This study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection. (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 122.2 (pelleting) Protein markers EV: TF/ CD63/ Flotillin/ PrP/ HSP70 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 122.2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ PrP/ TF/ Flotillin ELISA Antibody details provided? No Detected EV-associated proteins PrP/ TF/ Flotillin Characterization: Particle analysis TRPS EM EM-type transmission EM

	EV140065	2/2	Homo sapiens	Blood plasma	(d)(U)C	Cheng L	2014	33%
Study summary Full title Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood All authors Cheng L, Sharples RA, Scicluna BJ, Hill AF Journal J Extracell Vesicles Abstract INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed (show more...)INTRODUCTION: microRNA (miRNA) are small non-coding RNA species that are transcriptionally processed in the host cell and released extracellularly into the bloodstream. Normally involved in post-transcriptional gene silencing, the deregulation of miRNA has been shown to influence pathogenesis of a number of diseases. BACKGROUND: Next-generation deep sequencing (NGS) has provided the ability to profile miRNA in biological fluids making this approach a viable screening tool to detect miRNA biomarkers. However, collection and handling procedures of blood needs to be greatly improved for miRNA analysis in order to reliably detect differences between healthy and disease patients. Furthermore, ribonucleases present in blood can degrade RNA upon collection rendering extracellular miRNA at risk of degradation. These factors have consequently decreased sensitivity and specificity of miRNA biomarker assays. METHODS: Here, we use NGS to profile miRNA in various blood components and identify differences in profiles within peripheral blood compared to cell-free plasma or serum and extracellular vesicles known as exosomes. We also analyse and compare the miRNA content in exosomes prepared by ultracentrifugation methods and commercial exosome isolation kits including treating samples with RNaseA. CONCLUSION: This study demonstrates that exosomal RNA is protected by RNaseA treatment and that exosomes provide a consistent source of miRNA for disease biomarker detection. (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 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 122.2 (pelleting) Protein markers EV: TF/ CD63/ Flotillin/ PrP/ HSP70 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 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 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 122.2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ HSP70/ PrP/ TF/ Flotillin ELISA Antibody details provided? No Detected EV-associated proteins PrP/ TF/ Flotillin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV140062	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Bronisz A	2014	33%
Study summary Full title Extracellular vesicles modulate the glioblastoma microenvironment via a tumor suppression signaling network directed by miR-1 All authors Bronisz A, Wang Y, Nowicki MO, Peruzzi P, Ansari KI, Ogawa D, Balaj L, De Rienzo G, Mineo M, Nakano I, Ostrowski MC, Hochberg F, Weissleder R, Lawler SE, Chiocca EA, Godlewski J Journal Cancer Res Abstract Extracellular vesicles have emerged as important mediators of intercellular communication in cancer, (show more...)Extracellular vesicles have emerged as important mediators of intercellular communication in cancer, including by conveying tumor-promoting microRNAs between cells, but their regulation is poorly understood. In this study, we report the findings of a comparative microRNA profiling and functional analysis in human glioblastoma that identifies miR-1 as an orchestrator of extracellular vesicle function and glioblastoma growth and invasion. Ectopic expression of miR-1 in glioblastoma cells blocked in vivo growth, neovascularization, and invasiveness. These effects were associated with a role for miR-1 in intercellular communication in the microenvironment mediated by extracellular vesicles released by cancer stem-like glioblastoma cells. An extracellular vesicle-dependent phenotype defined by glioblastoma invasion, neurosphere growth, and endothelial tube formation was mitigated by loading miR-1 into glioblastoma-derived extracellular vesicles. Protein cargo in extracellular vesicles was characterized to learn how miR-1 directed extracellular vesicle function. The mRNA encoding Annexin A2 (ANXA2), one of the most abundant proteins in glioblastoma-derived extracellular vesicles, was found to be a direct target of miR-1 control. In addition, extracellular vesicle-derived miR-1 along with other ANXA2 extracellular vesicle networking partners targeted multiple pro-oncogenic signals in cells within the glioblastoma microenvironment. Together, our results showed how extracellular vesicle signaling promotes the malignant character of glioblastoma and how ectopic expression of miR-1 can mitigate this character, with possible implications for how to develop a unique miRNA-based therapy for glioblastoma management. (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: YWHAZ/ CD63/ Annexin2/ FASN/ CD9 non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ Annexin2/ FASN/ YWHAZ ELISA Antibody details provided? No Detected EV-associated proteins Annexin2/ FASN/ YWHAZ Characterization: Particle analysis NTA EM EM-type immune EM EM protein CD63 Image type Close-up, Wide-field

	EV140022	1/1	Homo sapiens	NAY	(d)(U)C	Boelens MC	2014	33%
Study summary Full title Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways All authors Boelens MC, Wu TJ, Nabet BY, Xu B, Qiu Y, Yoon T, Azzam DJ, Twyman-Saint Victor C, Wiemann BZ, Ishwaran H, Ter Brugge PJ, Jonkers J, Slingerland J, Minn AJ Journal Cell Abstract Stromal communication with cancer cells can influence treatment response. We show that stromal and b (show more...)Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely noncoding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent antiviral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine antiviral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy-resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of antiviral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate crosstalk with BrCa cells by utilizing exosomes to instigate antiviral signaling. This expands BrCa subpopulations adept at resisting therapy and reinitiating tumor growth. (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: TSG101/ CD81/ Flotilin1/ Annexin5/ ICAM1/ EpCam/ Beta-actin non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ Flotilin1/ TSG101/ ICAM1/ EpCam/ Annexin5/ Beta-actin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins ICAM1/ EpCam/ Annexin5/ Beta-actin Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV140061	1/1	Homo sapiens	NAY	(d)(U)C	Bian S	2014	33%
Study summary Full title Extracellular vesicles derived from human bone marrow mesenchymal stem cells promote angiogenesis in a rat myocardial infarction model All authors Bian S, Zhang L, Duan L, Wang X, Min Y, Yu H Journal J Mol Med (Berl) Abstract Mesenchymal stem cells (MSCs) have been increasingly tested experimentally and clinically for cardia (show more...)Mesenchymal stem cells (MSCs) have been increasingly tested experimentally and clinically for cardiac repair. However, the underlying mechanisms remain controversial due to the poor viability and considerable death of the engrafted cells in the infracted myocardium. Recent reports have suggested that extracellular vesicles (EVs) released by MSCs have angiogenesis-promoting activity; however, the therapeutic effect of MSC-EVs on an ischemic heart is unclear. In the present study, we reported that MSCs could release a large quantity of EVs around 100 nm in diameter upon hypoxia stimulation though the majority of the cells had not experienced apoptosis. MSC-EVs could be promptly uptaken by human umbilical vein endothelial cells, and the internalization resulted in dose-dependent enhancement of in vitro proliferation, migration, and tube formation of endothelial cells. Using an acute myocardial infarction rat model, we found that intramyocardial injection of MSC-EVs markedly enhanced blood flow recovery, in accordance with reduced infarct size and preserved cardiac systolic and diastolic performance compared to those treated with PBS. These data suggest that like MSCs, MSC-EVs could also protect cardiac tissue from ischemic injury at least by means of promoting blood vessel formation, though further detailed investigations should be performed to define the functionality of MSC-EVs.KEY MESSAGES: MSCs released extracellular vesicles (EVs) upon hypoxia stimulation. MSC-EVs were a mixture of microvesicles and exosomes. MSC-EVs could be promptly uptaken by human umbilical vein endothelial cells. MSC-EVs promoted neoangiogenesis in vitro and in vivo. MSC-EVs preserved cardiac performance in an AMI model. (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 Protein markers EV: CD81/ CD63 non-EV: CD29/ CD73/ CD44/ CD45/ CD31/ Albumin Proteomics no TEM measurements 106+-31 (47-180) Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Detected contaminants Albumin/ "CD29/ CD44/ CD73/ CD31/ CD45" Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis EM EM-type transmission EM Image type Close-up Report size (nm) 106+-31 (47-180)

	EV140060	1/1	Rattus norvegicus/rattus	Blood plasma	ExoQuick	Beninson LA	2014	33%
Study summary Full title Acute stressor exposure modifies plasma exosome-associated heat shock protein 72 (Hsp72) and microRNA (miR-142-5p and miR-203) All authors Beninson LA, Brown PN, Loughridge AB, Saludes JP, Maslanik T, Hills AK, Woodworth T, Craig W, Yin H, Fleshner M Journal PLoS One Abstract Exosomes, biologically active nanoparticles (40-100 nm) released by hematopoietic and non-hematopoie (show more...)Exosomes, biologically active nanoparticles (40-100 nm) released by hematopoietic and non-hematopoietic cells, contain a variety of proteins and small, non-coding RNA known as microRNA (miRNA). Exposure to various pathogens and disease states modifies the composition and function of exosomes, but there are no studies examining in vivo exosomal changes evoked by the acute stress response. The present study reveals that exposing male Fisher 344 rats to an acute stressor modulates the protein and miRNA profile of circulating plasma exosomes, specifically increasing surface heat shock protein 72 (Hsp72) and decreasing miR-142-5p and -203. The selected miRNAs and Hsp72 are associated with immunomodulatory functions and are likely a critical component of stress-evoked modulation of immunity. Further, we demonstrate that some of these stress-induced modifications in plasma exosomes are mediated by sympathetic nervous system (SNS) activation of alpha-1 adrenergic receptors (ADRs), since drug-mediated blockade of the receptors significantly attenuates the stress-induced modifications of exosomal Hsp72 and miR-142-5p. Together, these findings demonstrate that activation of the acute stress response modifies the proteomic and miRNA profile of exosomes released into the circulation. (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 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 Protein markers EV: Rab5a/ HSP72/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Blood plasma Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSP72/ Rab5a ELISA Antibody details provided? No Detected EV-associated proteins CD63/ HSP72/ Rab5a 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

	EV140121	1/2	Homo sapiens	NAY	(d)(U)C	Atay S	2014	33%
Study summary Full title Oncogenic KIT-containing exosomes increase gastrointestinal stromal tumor cell invasion All authors Atay S, Banskota S, Crow J, Sethi G, Rink L, Godwin AK Journal Proc Natl Acad Sci U S A Abstract During tumor development, constant interplay occurs between tumor cells and surrounding stromal cell (show more...)During tumor development, constant interplay occurs between tumor cells and surrounding stromal cells. We report evidence that gastrointestinal stromal tumor (GIST) cells invade the interstitial stroma through the release of the oncogenic protein tyrosine kinase (KIT)-containing exosomes, which triggers the phenotypic conversion of progenitor smooth muscle cells to tumor-promoting cells. These recipient cells display morphologic changes and acquire tumor-associated phenotypes, including enhanced adhesion to extracellular matrix proteins, activation of intracellular pathways downstream of KIT, expression of Interstitial Cell of Cajal-like markers, and release of various matrix metalloproteinases (MMPs), particularly MMP1. This report shows stimulation of MMP1 production by stromal cells via uptake of tumor-derived exosomes, which leads to tumor cell invasion. Exosomes derived from GIST patients but not healthy donors show enhanced MMP1 secretion by smooth muscle cells and tumor cell invasion, whereas selective blocking of exosome-mediated MMP1 secretion decreases tumor invasiveness. Our study indicates that exosome release and subsequent MMP1 induction creates a positive feedback mechanism established between tumor and stromal cells that drives GIST development and offers unique insights for potential therapeutic strategies to block GIST progression and metastatic spread. (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: TSG101/ Alix/ CD63/ Flotillin/ Annexin1/ CD9 non-EV: Beta-actin/ Cell organelle protein Proteomics no TEM measurements 133+-13;183+-27 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 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ CD63/ CD9/ TSG101/ Annexin1/ Flotillin Detected contaminants Cell organelle protein/ Beta-actin ELISA Antibody details provided? No Detected EV-associated proteins Annexin1/ Flotillin Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field Report size (nm) 133+-13;183+-27

	EV140057	1/2	Mus musculus	NAY	(d)(U)C Filtration	Aswad H	2014	33%
Study summary Full title Exosomes participate in the alteration of muscle homeostasis during lipid-induced insulin resistance in mice All authors Aswad H, Forterre A, Wiklander OP, Vial G, Danty-Berger E, Jalabert A, Lamazière A, Meugnier E, Pesenti S, Ott C, Chikh K, El-Andaloussi S, Vidal H, Lefai E, Rieusset J, Rome S Journal Diabetologia Abstract AIMS/HYPOTHESIS: Exosomes released from cells can transfer both functional proteins and RNAs between (show more...)AIMS/HYPOTHESIS: Exosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route. METHODS: Exosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA). RESULTS: HP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues. CONCLUSIONS/INTERPRETATION: Exosomes act as paracrine-like signals and modify muscle homeostasis during high-fat diets. (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 Adj. k-factor 157.1 (pelleting) / 157.1 (washing) Protein markers EV: Alix/ TSG101 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 Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 502Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 25 Wash: Rotor Type 50.2Ti Wash: adjusted k-factor 157.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140057	2/2	Mus musculus	Mouse muscle	(d)(U)C Filtration	Aswad H	2014	33%
Study summary Full title Exosomes participate in the alteration of muscle homeostasis during lipid-induced insulin resistance in mice All authors Aswad H, Forterre A, Wiklander OP, Vial G, Danty-Berger E, Jalabert A, Lamazière A, Meugnier E, Pesenti S, Ott C, Chikh K, El-Andaloussi S, Vidal H, Lefai E, Rieusset J, Rome S Journal Diabetologia Abstract AIMS/HYPOTHESIS: Exosomes released from cells can transfer both functional proteins and RNAs between (show more...)AIMS/HYPOTHESIS: Exosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route. METHODS: Exosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA). RESULTS: HP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues. CONCLUSIONS/INTERPRETATION: Exosomes act as paracrine-like signals and modify muscle homeostasis during high-fat diets. (hide) EV-METRIC 33% (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 Mouse muscle 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 157.1 (pelleting) / 157.1 (washing) Protein markers EV: Alix/ TSG101 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Mouse muscle Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 502Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 25 Wash: Rotor Type 50.2Ti Wash: adjusted k-factor 157.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140021	1/1	Homo sapiens	NAY	(d)(U)C	Amorim M	2014	33%
Study summary Full title The overexpression of a single oncogene (ERBB2/HER2) alters the proteomic landscape of extracellular vesicles All authors Amorim M, Fernandes G, Oliveira P, Martins-de-Souza D, Dias-Neto E, Nunes D Journal Proteomics Abstract ERBB2/HER2 amplification activates signaling cascades that lead to a tumor cell phenotype. However, (show more...)ERBB2/HER2 amplification activates signaling cascades that lead to a tumor cell phenotype. However, despite its remarkable importance in oncology, the consequences of HER2 amplification over the extracellular vesicles (EVs) content have not yet been investigated. Here, we isolated EVs secreted by HB4a, a mammary luminal epithelial cell line and C5.2, its HER2-overexpressing clone. We isolated two EV sets (20 and 100 K) by ultracentrifugation and used electron microscopy and nanoparticle tracking analysis for their morphological characterization. We employed GeLC-MS/MS combined with isotope-coded protein labeling to evaluate cell-derived proteins and LC-MS/MS label free spectral counting to quantify the EVs proteome. We found higher HER2 levels in both C5.2-derived EVs when compared with C5.2 cells, suggesting its preferential shuttling. Proteins capable of inducing malignant transformation are enriched in both C5.2 EV subsets, including two HER2-related proteins involved in cell motility and invasion, cofilin and CD44. MetaCore™ analysis indicated an enrichment of cell adhesion and cytoskeleton-remodeling pathways in C5.2 EVs, as well as proteins related to HER2 signaling, such as sphingosine-1-phosphate pathway. Together, our data indicate that in terms of protein content, distinct vesicle sets reinforce and complement each other. Our results also suggest that HER2-upregulated proteins from EVs may be relevant for cellular malignancy and can be potential biomarkers for HER2(+) cancer patients. (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 Protein markers EV: HSP70/ Rab27B/ Flotilin1 non-EV: 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 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Flotilin1/ HSP70/ Rab27B ELISA Antibody details provided? No Detected EV-associated proteins Rab27B Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140019	1/1	Sus scrofa	NAY	(d)(U)C Filtration UF	Aboul Naga SH	2014	33%
Study summary Full title Intracellular pathways following uptake of bevacizumab in RPE cells All authors Aboul Naga SH, Dithmer M, Chitadze G, Kabelitz D, Lucius R, Roider J, Klettner A Journal Exp Eye Res Abstract The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular dise (show more...)The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular diseases, most commonly in age-related macular degeneration and diabetic macular edema. Bevacizumab is able to penetrate the retina and is found in the choroid after intravitreal injection in a time dependent manner. It has previously been shown to be taken up by the retinal pigment epithelium (RPE). In this study, we have investigated the intracellular pathway following uptake of bevacizumab in RPE cells, tested both in primary porcine RPE cells and in the human cell line ARPE19. Bevacizumab displays a characteristic, time-dependent pattern of intracellular distribution, as detected by immunofluorescence and pulse chase experiments. In both primary cells and the cell line, intracellular bevacizumab can be found after seven days, as detected by immunofluorescence and Western blotting. Immediately after application, bevacizumab partially colocalizes with Rab5, indicating some uptake in early endosomes. Intracellularly, bevacizumab is detected in the cytoskeletal fraction, aligning with actin filaments, as revealed by subcellular fractioning and immunofluorescence. Bevacizumab seems to travel along actin filaments by myosin7a, as determined by triple staining immunofluorescence. Interestingly, over a period of seven days, bevacizumab seems to accumulate in certain storage areas, as observed by immunofluorescence. Furthermore, results obtained with immunocytochemistry, Western blotting and flow cytometry indicate that bevacizumab may be released from the RPE cells via exosomes. In conclusion, bevacizumab is taken up by and transported in the retinal pigment epithelial cells in a characteristic, time-dependent manner, where it seems to move along actin filaments by myosin7a and seem to be partially released from the cells via exosomes. (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 UF Protein markers EV: TSG101/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Sus scrofa Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ TSG101 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140290	2/3	Homo sapiens	NAY	(d)(U)C UF	van der Mijn JC	2014	29%
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 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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: non-EV: Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 Characterization: Particle analysis None

	EV140258	3/3	Mus musculus	Serum	(d)(U)C DG Filtration	Ridder K	2014	29%
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 29% (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 Serum 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: non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 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 21 Orientation Top-down Rotor type SW40 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis None

	EV140129	1/2	Homo sapiens	NAY	(d)(U)C	Ma X	2014	29%
Study summary Full title Essential role for TrpC5-containing extracellular vesicles in breast cancer with chemotherapeutic resistance All authors Ma X, Chen Z, Hua D, He D, Wang L, Zhang P, Wang J, Cai Y, Gao C, Zhang X, Zhang F, Wang T, Hong T, Jin L, Qi X, Chen S, Gu X, Yang D, Pan Q, Zhu Y, Chen Y, Chen D, Jiang L, Han X, Zhang Y, Jin J, Yao X Journal Proc Natl Acad Sci U S A Abstract A critical challenge for chemotherapy is the development of chemoresistance in breast cancer. Howeve (show more...)A critical challenge for chemotherapy is the development of chemoresistance in breast cancer. However, the underlying mechanisms and validated predictors remain unclear. Extracellular vesicles (EVs) have gained attention as potential means for cancer cells to share intracellular contents. In adriamycin-resistant human breast cancer cells (MCF-7/ADM), we analyzed the role of transient receptor potential channel 5 (TrpC5) in EV formation and transfer as well as the diagnostic implications. Up-regulated TrpC5, accumulated in EVs, is responsible for EV formation and trapping of adriamycin (ADM) in EVs. EV-mediated intercellular transfer of TrpC5 allowed recipient cells to acquire TrpC5, consequently stimulating multidrug efflux transporter P-glycoprotein production through a Ca(2+)- and activated T-cells isoform c3-mediated mechanism and thus, conferring chemoresistance on nonresistant cells. TrpC5-containing circulating EVs were detected in nude mice bearing MCF-7/ADM tumor xenografts, and the level was lower after TrpC5-siRNA treatment. In breast cancer patients who underwent chemotherapy, TrpC5 expression in the tumor was significantly higher in patients with progressive or stable disease than in patients with a partial or complete response. TrpC5-containing circulating EVs were found in peripheral blood from patients who underwent chemotherapy but not patients without chemotherapy. Taken together, we found that TrpC5-containing circulating EVs may transfer chemoresistance property to nonchemoresistant recipient cells. It may be worthwhile to further explore the potential of using TrpC5-containing EVs as a diagnostic biomarker for chemoresistant breast cancer. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 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) 120 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis EM EM-type transmission EM/ immune EM/ scanning EM EM protein Adriamycin; TrpC5; Flotillin2 Image type Close-up, Wide-field Report size (nm) Not reported

	EV140252	1/1	Homo sapiens	NAY	(d)(U)C Filtration filter	L?rincz ÁM	2014	29%
Study summary Full title Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes All authors Lorincz ÁM, Timár CI, Marosvári KA, Veres DS, Otrokocsi L, Kittel Á, Ligeti E Journal J Extracell Vesicles Abstract AIM: To carry out a systematic study on the effect of different storage conditions on the number as (show more...)AIM: To carry out a systematic study on the effect of different storage conditions on the number as well as the physical and functional properties of antibacterial extracellular vesicles (EVs) derived from human neutrophilic granulocytes. METHODS: Production of EVs with antibacterial properties was initiated by opsonized Zymosan A particles. The number of released fluorescent EVs was determined by flow cytometry following careful calibration. Physical properties and size of EVs were investigated by flow cytometry, dynamic light scattering and electron microscopy. Functional properties of EVs were tested by bacterial survival assay. RESULTS: Storage at +20°C or +4°C resulted in a significant decrease of EV number and antibacterial effect after 1 day. Storage at -20°C did not influence the EV number up to 28 days, but induced a shift in EV size and almost complete loss of antibacterial function by 28 days. Storage at -80°C had no significant effect either on EV number or size and allowed partial preservation of the antibacterial function up to 28 days. Snap-freezing did not improve the results, whereas the widely used cryoprotectants induced EV lysis. CONCLUSION: Storage significantly alters both the physical and functional properties of EVs even if the number of EVs stays constant. If storage is needed, EVs should be kept at -80°C, preferably not longer than 7 days. For functional tests, freshly prepared EVs are recommended. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles 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 filter Adj. k-factor 373.5 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Technical 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 Pelleting performed Yes Pelleting: time(min) 10 Pelleting: rotor type Hermle Z216MK Pelleting: adjusted k-factor 373.5 Other Name other separation method filter Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140105	1/1	Mycobacterium tuberculosis	Bacteria	(d)(U)C DG Filtration UF	Prados-Rosales R	2014	29%
Study summary Full title Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition All authors Prados-Rosales R, Weinrick BC, Piqué DG, Jacobs WR Jr, Casadevall A, Rodriguez GM Journal J Bacteriol Abstract Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the im (show more...)Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host. (hide) EV-METRIC 29% (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 Bacteria Sample origin NAY Focus vesicles Membrane(-derived) vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration UF Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Mycobacterium tuberculosis Sample Type Bacteria Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Lowest density fraction 5 Highest density fraction 30 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140235	1/1	Homo sapiens	NAY	(d)(U)C	Xu JF	2014	29%
Study summary Full title Altered MicroRNA Expression Profile in Exosomes during Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells All authors Xu JF, Yang GH, Pan XH, Zhang SJ, Zhao C, Qiu BS, Gu HF, Hong JF, Cao L, Chen Y, Xia B, Bi Q, Wang YP Journal PLoS One Abstract The physiological role of microRNAs (miRNAs) in osteoblast differentiation remains elusive. Exosomal (show more...)The physiological role of microRNAs (miRNAs) in osteoblast differentiation remains elusive. Exosomal miRNAs isolated from human bone marrow-derived mesenchymal stem cells (BMSCs) culture were profiled using miRNA arrays containing probes for 894 human matured miRNAs. Seventy-nine miRNAs (?8.84%) could be detected in exosomes isolated from BMSC culture supernatants when normalized to endogenous control genes RNU44. Among them, nine exosomal miRNAs were up regulated and 4 miRNAs were under regulated significantly (Relative fold>2, p<0.05) when compared with the values at 0 day with maximum changes at 1 to 7 days. Five miRNAs (miR-199b, miR-218, miR-148a, miR-135b, and miR-221) were further validated and differentially expressed in the individual exosomal samples from hBMSCs cultured at different time points. Bioinformatic analysis by DIANA-mirPath demonstrated that RNA degradation, mRNA surveillance pathway, Wnt signaling pathway, RNA transport were the most prominent pathways enriched in quantiles with differential exosomal miRNA patterns related to osteogenic differentiation. These data demonstrated exosomal miRNA is a regulator of osteoblast differentiation. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 396.8 (pelleting) / 167.3 (washing) Protein markers EV: CD63/ CD86/ MHC2/ CD54/ MHC1 non-EV: Proteomics no 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 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 110 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 396.8 Wash: Rotor Type SW60 Wash: adjusted k-factor 167.3 Western Blot Antibody details provided? No Detected EV-associated proteins MHC2/ MHC1/ CD86/ CD54 ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ MHC1/ CD86/ CD54 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis None

	EV140393	1/1	Mus musculus	NAY	(d)(U)C Filtration UF	Sobo-Vujanovic A	2014	29%
Study summary Full title Dendritic-cell exosomes cross-present Toll-like receptor-ligands and activate bystander dendritic cells All authors Sobo-Vujanovic A, Munich S, Vujanovic NL Journal Cell Immunol Abstract Dendritic cells (DCs) are the major sentinel, antigen-presenting and regulatory components of the im (show more...)Dendritic cells (DCs) are the major sentinel, antigen-presenting and regulatory components of the immune system. One of the central DC functions is to rapidly sense and alert host immune system of a pathogen invasion. In the present study, we investigated the role of DC exosomes (DCex) in this sentinel function. We demonstrated that DCex could bind bacterial Toll-like-receptor ligands (TLR-Ls), and acquire their ability to strongly activate bystander DCs. Consequently, bystander DCs enhance the expression of transmembrane tumor necrosis factor, secretion of proinflammatory cytokines and cross-talk with natural killer cells leading to the elevated secretion of IFN?. These findings newly show that DCex can bind and cross-present TLR-Ls to innate-immunity effector cells, and indicate a potent mechanism to systemically alert the host immune system of pathogen invasion. They also suggest a potential novel strategy to generate effective vaccines by binding TLR-L-immune adjuvants to DCex. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 126 (pelleting) Protein markers EV: CD14/ MHC2/ TLR4 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type 90Ti Pelleting: adjusted k-factor 126 Filtration steps 0.45µm > x > 0.22µm, Western Blot Antibody details provided? No Detected EV-associated proteins MHC2/ CD14/ TLR4 ELISA Antibody details provided? No Detected EV-associated proteins MHC2/ CD14/ TLR4 Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis None

	EV140283	2/2	Homo sapiens	Ascites	(d)(U)C DG UF	Shender VO	2014	29%
Study summary Full title Proteome-metabolome profiling of ovarian cancer ascites reveals novel components involved in intercellular communication All authors Shender VO, Pavlyukov MS, Ziganshin RH, Arapidi GP, Kovalchuk SI, Anikanov NA, Altukhov IA, Alexeev DG, Butenko IO, Shavarda AL, Khomyakova EB, Evtushenko E, Ashrafyan LA, Antonova IB, Kuznetcov IN, Gorbachev AY, Shakhparonov MI, Govorun VM Journal Mol Cell Proteomics Abstract Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secret (show more...)Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication. (hide) EV-METRIC 29% (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 Ascites Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG UF Adj. k-factor 159.6 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Omics Sample Species Homo sapiens Sample Type Ascites Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 60Ti Pelleting: adjusted k-factor 159.6 Density gradient Lowest density fraction 15 Highest density fraction 48 Orientation Bottom-up Speed (g) 200000 Characterization: Particle analysis NTA

	EV140282	1/2	Mus musculus	NAY	(d)(U)C DC Filtration	Saunderson SC	2014	29%
Study summary Full title CD169 mediates the capture of exosomes in spleen and lymph node All authors Saunderson SC, Dunn AC, Crocker PR, McLellan AD Journal Blood Abstract Exosomes are lipid nanovesicles released following fusion of the endosoma limiting membrane with the (show more...)Exosomes are lipid nanovesicles released following fusion of the endosoma limiting membrane with the plasma membrane; however, their fate in lymphoid organs after their release remains controversial. We determined that sialoadhesin (CD169; Siglec-1) is required for the capture of B cell-derived exosomes via their surface-expressed ?2,3-linked sialic acids. Exosome-capturing macrophages were present in the marginal zone of the spleen and in the subcapsular sinus of the lymph node. In vitro assays performed on spleen and lymph node sections confirmed that exosome binding to CD169 was not solely due to preferential fluid flow to these areas. Although the circulation half-life of exosomes in blood of wild-type and CD169(-/-) mice was similar, exosomes displayed altered distribution in CD169(-/-) mice, with exosomes freely accessing the outer marginal zone rim of SIGN-R1(+) macrophages and F4/80(+) red pulp macrophages. In the lymph node, exosomes were not retained in the subcapsular sinus of CD169(-/-) mice but penetrated deeper into the paracortex. Interestingly, CD169(-/-) mice demonstrated an enhanced response to antigen-pulsed exosomes. This is the first report of a role for CD169 in the capture of exosomes and its potential to mediate the immune response to exosomal antigen. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration Protein markers EV: MHC2/ CD9 non-EV: a2,3-sialic acid/ CD21/ CD24/ CD19/ a2,6-sialic acid/ Ig Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 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 MHC2 ELISA Antibody details provided? No Detected EV-associated proteins CD9/ MHC2 Detected contaminants "CD19/ CD21/ CD24/ Ig/ a2,3-sialic acid/ a2,6-sialic acid" Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV140269	2/3	Homo sapiens	Brain tissue	(d)(U)C DG Filtration	Saman S	2014	29%
Study summary Full title Proteins recruited to exosomes by tau overexpression implicate novel cellular mechanisms linking tau secretion with Alzheimer's disease All authors Saman S, Lee NC, Inoyo I, Jin J, Li Z, Doyle T, McKee AC, Hall GF Journal J Alzheimers Dis Abstract Tau misprocessing to form aggregates and other toxic species has emerged as a major feature in our d (show more...)Tau misprocessing to form aggregates and other toxic species has emerged as a major feature in our developing understanding of the etiology and pathogenesis of Alzheimer's disease (AD). The significance of tau misprocessing in AD has been further emphasized by recent studies showing that tau can be secreted from neurons via exosomes and may itself be an important agent in the spreading of neurofibrillary lesions within the brain. Tau secretion occurs most readily under disease-associated conditions in cellular models, suggesting that cellular changes responsible for secretion, possibly including tau oligomerization, could play a key role in the propagation of neurofibrillary lesions in neurodegenerative disease. Here we show that overexpression of 4R0N human tau in neuroblastoma cells recruits mitochondrial and axonogenesis-associated proteins relevant to neurodegeneration into the exosomal secretion pathway via distinct mechanisms. The recruitment of mitochondrial proteins appears to be linked to autophagy disruption (exophagy) in multiple neurodegenerative conditions but has few known direct links to AD and tau. By contrast, the involvement of synaptic plasticity and axonogenesis markers is highly specific to both tau and AD and may be relevant to the reactivation of developmental programs involving tau in AD and the recently demonstrated ability of secreted tau to establish tissue distribution gradients in CNS neuropil. We also found a highly significant correlation between genes that are significantly downregulated in multiple forms of AD and proteins that have been recruited to exosomes by tau, which we interpret as strong evidence for the central involvement of tau secretion in AD cytopathogenesis. Our results suggest that multiple cellular mechanisms may link tau secretion to both toxicity and neurofibrillary lesion spreading in AD and other tauopathies. (hide) EV-METRIC 29% (29th 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 Brain tissue 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: non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Brain 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) 70 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Characterization: Protein analysis Characterization: Particle analysis None

	EV140269	3/3	Homo sapiens	"Cerebrospinal fluid"	(d)(U)C DG	Saman S	2014	29%
Study summary Full title Proteins recruited to exosomes by tau overexpression implicate novel cellular mechanisms linking tau secretion with Alzheimer's disease All authors Saman S, Lee NC, Inoyo I, Jin J, Li Z, Doyle T, McKee AC, Hall GF Journal J Alzheimers Dis Abstract Tau misprocessing to form aggregates and other toxic species has emerged as a major feature in our d (show more...)Tau misprocessing to form aggregates and other toxic species has emerged as a major feature in our developing understanding of the etiology and pathogenesis of Alzheimer's disease (AD). The significance of tau misprocessing in AD has been further emphasized by recent studies showing that tau can be secreted from neurons via exosomes and may itself be an important agent in the spreading of neurofibrillary lesions within the brain. Tau secretion occurs most readily under disease-associated conditions in cellular models, suggesting that cellular changes responsible for secretion, possibly including tau oligomerization, could play a key role in the propagation of neurofibrillary lesions in neurodegenerative disease. Here we show that overexpression of 4R0N human tau in neuroblastoma cells recruits mitochondrial and axonogenesis-associated proteins relevant to neurodegeneration into the exosomal secretion pathway via distinct mechanisms. The recruitment of mitochondrial proteins appears to be linked to autophagy disruption (exophagy) in multiple neurodegenerative conditions but has few known direct links to AD and tau. By contrast, the involvement of synaptic plasticity and axonogenesis markers is highly specific to both tau and AD and may be relevant to the reactivation of developmental programs involving tau in AD and the recently demonstrated ability of secreted tau to establish tissue distribution gradients in CNS neuropil. We also found a highly significant correlation between genes that are significantly downregulated in multiple forms of AD and proteins that have been recruited to exosomes by tau, which we interpret as strong evidence for the central involvement of tau secretion in AD cytopathogenesis. Our results suggest that multiple cellular mechanisms may link tau secretion to both toxicity and neurofibrillary lesion spreading in AD and other tauopathies. (hide) EV-METRIC 29% (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 "Cerebrospinal 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 DG Protein markers EV: non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type "Cerebrospinal fluid" Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2 Orientation Top-down Characterization: Protein analysis Characterization: Particle analysis None

	EV140211	1/1	Homo sapiens	NAY	(d)(U)C DG Filtration	Rupert DL	2014	29%
Study summary Full title Determination of exosome concentration in solution using surface plasmon resonance spectroscopy All authors Rupert DL, Lässer C, Eldh M, Block S, Zhdanov VP, Lotvall JO, Bally M, Höök F Journal Anal Chem Abstract Exosomes are cell-secreted nanometer-sized extracellular vesicles that have been reported to play an (show more...)Exosomes are cell-secreted nanometer-sized extracellular vesicles that have been reported to play an important role in intercellular communication. They are also considered potential diagnostic markers for various health disorders, and intense investigations are presently directed toward their use as carriers in drug-delivery and gene-therapy applications. This has generated a growing need for sensitive methods capable of accurately and specifically determining the concentration of exosomes in complex biological fluids. Here, we explore the use of label-free surface-based sensing with surface plasmon resonance (SPR) read-out to determine the concentration of exosomes in solution. Human mast cell secreted exosomes carrying the tetraspanin membrane protein CD63 were analyzed by measuring their diffusion-limited binding rate to an SPR sensor surface functionalized with anti-CD63 antibodies. The concentration of suspended exosomes was determined by first converting the SPR response into the surface-bound mass. The increase in mass uptake over time was then related to the exosome concentration in solution using a formalism describing diffusion-limited binding under controlled flow conditions. The proposed quantification method is based on a calibration and control measurements performed with proteins and synthetic lipid vesicles and takes into account (i) the influence of the broad size distribution of the exosomes on the surface coverage, (ii) the fact that their size is comparable to the ?150 nm probing depth of SPR, and (iii) possible deformation of exosomes upon adsorption. Under those considerations, the accuracy of the concentration determination was estimated to be better than ±50% and significantly improve if the exosome deformation is negligible. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.24-1.31 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Density gradient Only used for validation of main results Yes Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis NTA

	EV140280	1/2	Mus musculus	NAY	(d)(U)C IAF	Rahman MJ	2014	29%
Study summary Full title Exosomes released by islet-derived mesenchymal stem cells trigger autoimmune responses in NOD mice All authors Rahman MJ, Regn D, Bashratyan R, Dai YD Journal Diabetes Abstract Exosomes (EXOs) are secreted, nano-sized membrane vesicles that contain potent immunostimulatory mat (show more...)Exosomes (EXOs) are secreted, nano-sized membrane vesicles that contain potent immunostimulatory materials. We have recently demonstrated that insulinoma-released EXOs can stimulate the autoimmune responses in nonobese diabetic (NOD) mice, a spontaneous disease model for type 1 diabetes. To investigate whether primary islet cells can produce EXOs, we isolated cells from the islet of Langerhans of NOD mice and cultured them in vitro. Interestingly, cultured islets release fibroblast-like, fast-replicating cells that express mesenchymal stem cell (MSC) markers, including CD105 and stem-cell antigen-1. These islet MSC-like cells release highly immunostimulatory EXOs that could activate autoreactive B and T cells endogenously primed in NOD mice. Serum EXO levels and EXO-induced interferon-? production were positively correlated with disease progression at the early prediabetic stage. Consistent with these observations, immunohistological analysis of pancreata showed that CD105(+) cells are restricted to the peri-islet area in normal islets but penetrate into the ?-cell area as lymphocyte infiltration occurs. Immunization with EXOs promoted expansion of transferred diabetogenic T cells and accelerated the effector T cell-mediated destruction of islets. Thus, EXOs could be the autoantigen carrier with potent adjuvant activities and may function as the autoimmune trigger in NOD mice. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C IAF Protein markers EV: CD81/ CD63 non-EV: Proteomics yes Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV140205	1/1	Homo sapiens	NAY	(d)(U)C DG Filtration	Philip R	2014	29%
Study summary Full title Claudin-7 promotes the epithelial-mesenchymal transition in human colorectal cancer All authors Philip R, Heiler S, Mu W, Büchler MW, Zöller M, Thuma F Journal Oncotarget Abstract In colorectal cancer (CoCa) EpCAM is frequently associated with claudin-7. There is evidence that tu (show more...)In colorectal cancer (CoCa) EpCAM is frequently associated with claudin-7. There is evidence that tumor-promoting EpCAM activities are modulated by the association with claudin-7. To support this hypothesis, claudin-7 was knocked-down (kd) in HT29 and SW948 cells. HT29-cld7kd and SW948-cld7kd cells display decreased anchorage-independent growth and the capacity for holoclone-, respectively, sphere-formation is reduced. Tumor growth is delayed and cld7kd cells poorly metastasize. In line with this, migratory and invasive potential of cld7kd clones is strongly impaired, migration being inhibited by anti-CD49c, but not anti-EpCAM, although motility is reduced in EpCAM siRNA-treated cells. This is due to claudin-7 recruiting EpCAM in glycolipid-enriched membrane fractions towards claudin-7-associated TACE and presenilin2, which cleave EpCAM. The cleaved intracellular domain, EpIC, promotes epithelial-mesenchymal transition (EMT)-associated transcription factor expression, which together with fibronectin and vimentin are reduced in claudin-7kd cells. But, uptake of HT29wt and SW948wt exosomes by the claudin-7kd lines sufficed for transcription factor upregulation and for restoring motility. Thus, claudin-7 contributes to motility and invasion and is required for recruiting EpCAM towards TACE/presenilin2. EpIC generation further supports motility by promoting a shift towards EMT. Notably, EMT features of cld7-competent metastatic CoCa cells can be transferred via exosomes to poorly metastatic cells. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: non-EV: Proteomics no EV density (g/ml) 1.12-1.17 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) 90 Density gradient Lowest density fraction 5 Highest density fraction 40 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis None

	EV140089	1/1	Mus musculus	NAY	(d)(U)C UF	Petersen KE	2014	29%
Study summary Full title A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM All authors Petersen KE, Manangon E, Hood JL, Wickline SA, Fernandez DP, Johnson WP, Gale BK Journal Anal Bioanal Chem Abstract Exosomes participate in cancer metastasis, but studying them presents unique challenges as a result (show more...)Exosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for label-free isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Protein markers EV: non-EV: Proteomics no Show all info Study aim Technical Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

	EV140087	1/2	Vitis Citrus paradisi	Plant material (grape, grapefruit)	(d)(U)C DG	Mu J	2014	29%
Study summary Full title Interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles All authors Mu J, Zhuang X, Wang Q, Jiang H, Deng ZB, Wang B, Zhang L, Kakar S, Jun Y, Miller D, Zhang HG Journal Mol Nutr Food Res Abstract SCOPE: Exosomes, small vesicles participating in intercellular communication, have been extensively (show more...)SCOPE: Exosomes, small vesicles participating in intercellular communication, have been extensively studied recently; however, the role of edible plant derived exosomes in interspecies communication has not been investigated. Here, we investigate the biological effects of edible plant derived exosome-like nanoparticles (EPDENs) on mammalian cells. METHODS AND RESULTS: In this study, exosome-like nanoparticles from four edible plants were isolated and characterized. We show that these EPDENs contain proteins, lipids, and microRNA. EPDENs are taken up by intestinal macrophages and stem cells. The results generated from EPDEN-transfected macrophages indicate that ginger EPDENs preferentially induce the expression of the antioxidation gene, heme oxygenase-1 and the anti-inflammatory cytokine, IL-10; whereas grapefruit, ginger, and carrot EPDENs promote activation of nuclear factor like (erythroid-derived 2). Furthermore, analysis of the intestines of canonical Wnt-reporter mice, i.e. B6.Cg-Tg(BAT-lacZ)3Picc/J mice, revealed that the numbers of ?-galactosidase(+) (?-Gal) intestinal crypts are increased, suggesting that EPDEN treatment of mice leads to Wnt-mediated activation of the TCF4 transcription machinery in the crypts. CONCLUSION: The data suggest a role for EPDEN-mediated interspecies communication by inducing expression of genes for anti-inflammation cytokines, antioxidation, and activation of Wnt signaling, which are crucial for maintaining intestinal homeostasis. (hide) EV-METRIC 29% (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 Plant material (grape, grapefruit) 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: non-EV: Proteomics no Show all info Study aim Function Sample Species Vitis / Citrus paradisi Sample Type Plant material (grape, grapefruit) 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) 90 Density gradient Lowest density fraction 8 Highest density fraction 60 Orientation Top-down Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140087	2/2	Zingiber officinale Daucus carota	Plant material (ginger, carrot)	(d)(U)C DG	Mu J	2014	29%
Study summary Full title Interspecies communication between plant and mouse gut host cells through edible plant derived exosome-like nanoparticles All authors Mu J, Zhuang X, Wang Q, Jiang H, Deng ZB, Wang B, Zhang L, Kakar S, Jun Y, Miller D, Zhang HG Journal Mol Nutr Food Res Abstract SCOPE: Exosomes, small vesicles participating in intercellular communication, have been extensively (show more...)SCOPE: Exosomes, small vesicles participating in intercellular communication, have been extensively studied recently; however, the role of edible plant derived exosomes in interspecies communication has not been investigated. Here, we investigate the biological effects of edible plant derived exosome-like nanoparticles (EPDENs) on mammalian cells. METHODS AND RESULTS: In this study, exosome-like nanoparticles from four edible plants were isolated and characterized. We show that these EPDENs contain proteins, lipids, and microRNA. EPDENs are taken up by intestinal macrophages and stem cells. The results generated from EPDEN-transfected macrophages indicate that ginger EPDENs preferentially induce the expression of the antioxidation gene, heme oxygenase-1 and the anti-inflammatory cytokine, IL-10; whereas grapefruit, ginger, and carrot EPDENs promote activation of nuclear factor like (erythroid-derived 2). Furthermore, analysis of the intestines of canonical Wnt-reporter mice, i.e. B6.Cg-Tg(BAT-lacZ)3Picc/J mice, revealed that the numbers of ?-galactosidase(+) (?-Gal) intestinal crypts are increased, suggesting that EPDEN treatment of mice leads to Wnt-mediated activation of the TCF4 transcription machinery in the crypts. CONCLUSION: The data suggest a role for EPDEN-mediated interspecies communication by inducing expression of genes for anti-inflammation cytokines, antioxidation, and activation of Wnt signaling, which are crucial for maintaining intestinal homeostasis. (hide) EV-METRIC 29% (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 Plant material (ginger, carrot) 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: non-EV: Proteomics no Show all info Study aim Function Sample Species Zingiber officinale / Daucus carota Sample Type Plant material (ginger, carrot) 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) 90 Density gradient Lowest density fraction 8 Highest density fraction 60 Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV140086	1/1	Mus musculus	NAY	(d)(U)C Filtration	Morishita M	2014	29%
Study summary Full title Quantitative Analysis of Tissue Distribution of the B16BL6-Derived Exosomes Using a Streptavidin-Lactadherin Fusion Protein and Iodine-125-Labeled Biotin Derivative After Intravenous Injection in Mice All authors Morishita M, Takahashi Y, Nishikawa M, Sano K, Kato K, Yamashita T, Imai T, Saji H, Takakura Y Journal J Pharm Sci Abstract We previously succeeded in the visualization of tissue distribution of B16BL6 cells-derived exosomes (show more...)We previously succeeded in the visualization of tissue distribution of B16BL6 cells-derived exosomes by labeling with Gaussia luciferase (gLuc)-LA, a fusion protein of gLuc (a reporter protein) and lactadherin (LA; an exosome-tropic protein). However, total amount of B16BL6-derived exosomes delivered to each organ could not be evaluated because of the reduction of luminescent signal from gLuc-LA. The aim of the present study was to quantitatively evaluate the tissue distribution of B16BL6-derived exosomes. To this end, we labeled B16BL6-derived exosomes with iodine-125 ((125) I) based on streptavidin (SAV)-biotin system. A plasmid vector encoding fusion protein, SAV-LA, was constructed, and B16BL6 cells were transfected with the plasmid to obtain SAV-LA-coupled exosomes. SAV-LA-coupled exosomes were incubated with (3-(125) I-iodobenzoyl) norbiotinamide ((125) I-IBB) to obtain (125) I-labeled B16BL6 exosomes. After intravenous injection of (125) I-labeled B16BL6 exosomes into mice, radioactivity quickly disappeared from the blood circulation. At 4 h, 28%, 1.6%, and 7% of the injected radioactivity/organ was detected in the liver, spleen, and lung, respectively. These results indicate that (125) I-labeling of exosomes using SAV-biotin system is a useful method to quantitatively evaluate the amount of exogenously administered exosomes delivered to each organ and that the liver is the major organ in the clearance of exogenously administered B16BL6-derived exosomes. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: non-EV: Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis NTA TRPS EM EM-type transmission EM Image type Close-up, Wide-field

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