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Results list Most used isolation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Isolation protocol	First author	Year	EV-METRIC
	EV150009	1/1	Homo sapiens	Placental perfusate	0.2 µm filter dUC	Dragovic RA	2015	67%
Study summary Full title Isolation of syncytiotrophoblast microvesicles and exosomes and their characterisation by multicolour flow cytometry and fluorescence Nanoparticle Tracking Analysis All authors Dragovic RA, Collett GP, Hole P, Ferguson DJ, Redman CW, Sargent IL, Tannetta DS Journal Methods Abstract The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesi (show more...)The human placenta releases multiple types and sizes of syncytiotrophoblast (STB) extracellular vesicles (EV) into the maternal circulation that exhibit diverse biological activities. The placental perfusion technique enables isolation of these STBEV, but conventional flow cytometry can only be used to phenotype EV down to ?300nm in size. Fluorescence Nanoparticle Tracking Analysis (fl-NTA) has the potential to phenotype EV down to ?50nm, thereby improving current characterisation techniques. The aims of this study were to prepare microvesicle and exosome enriched fractions from human placental perfusate (n=8) and improve fl-NTA STBEV detection. Differential centrifugation and filtration effectively removed contaminating red blood cells from fresh placental perfusates and pelleted a STB microvesicle (STBMV) fraction (10,000×g pellet - 10KP; NTA modal size 395±12nm), enriched for the STB marker placental alkaline phosphatase (PLAP) and a STB exosome (STBEX) fraction (150,000×g pellet - 150KP; NTA modal size 147±6nm), enriched for PLAP and exosome markers Alix and CD63. The PLAP positivity of standard 10KP and 150KP pools (four samples/pool), determined by immunobead depletion, was used to optimise fl-NTA camera settings. Individual 10KP and 150KP samples (n=8) were 54.5±5.7% (range 17.8-66.9%) and 30.6±5.6% (range 3.3-51.7%) PLAP positive, respectively. We have developed a reliable method for enriching STBMV and STBEX from placental perfusate. We also standardised fl-NTA settings and improved measurement of PLAP positive EV in STBMV. However, fl-NTA is not as sensitive as anti-PLAP Dynabead capture for STBEX detection, possibly due to STBEX having lower surface expression of PLAP. These important developments will facilitate more detailed studies of the role of STBMV and STBEX in normal and pathological pregnancies. (hide) EV-METRIC 67% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Placental perfusate Focus vesicles exosomes / microvesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Adj. k-factor 162 (pelleting) Protein markers EV: Alix/ CD63 non-EV: CD41/ CD235a/b/ CD45 Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Placental perfusate Isolation Method Differential ultracentrifugation 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: time(min) 120 Pelleting: rotor type TST28.39 Pelleting: adjusted k-factor 162.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63 Detected contaminants CD41/ CD235a/b/ CD45 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV150004	1/2	Homo sapiens	Cell culture supernatant	dUC Iodixanol-DG (valid.)	Clark DJ	2015	67%
Study summary Full title Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis All authors Clark DJ, Fondrie WE, Liao Z, Hanson PI, Fulton A, Mao L, Yang AJ Journal Anal Chem Abstract Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into t (show more...)Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as true exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression. (hide) EV-METRIC 67% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG (valid.) Protein markers EV: Alix/ CD63/ TSG101 non-EV: Cell organelle protein/ Beta-actin Proteomics yes EV density (g/ml) 1.14-1.19 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ TSG101 Detected contaminants Cell organelle protein/ Beta-actin Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV150008	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter Density cushion dUC Sucrose-DG (valid.)	Chan YK	2015	67%
Study summary Full title Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins All authors Chan YK, Zhang H, Liu P, Tsao SW, Lung ML, Mak NK, Ngok-Shun Wong R, Ying-Kit Yue P Journal Int J Cancer Abstract Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling (show more...)Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient and electron microscopy. We showed that the C666-1 exosomes (10 and 20 ?g/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (? 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1 and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. (hide) EV-METRIC 67% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Density cushion + dUC + Sucrose-DG (valid.) Adj. k-factor 156.9 (pelleting) Protein markers EV: CD63/ CD9 non-EV: Cell organelle protein Proteomics yes EV density (g/ml) 1.17-1.19 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.2 M Highest density fraction 2.5 M Orientation Top-down Rotor type 90Ti Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD9 Detected contaminants Cell organelle protein Characterization: Particle analysis TRPS

	EV150034	2/2	Homo sapiens	Blood plasma	SEC	de Menezes-Neto A	2015	57%
Study summary Full title Size-exclusion chromatography as a stand-alone methodology identifies novel markers in mass spectrometry analyses of plasma-derived vesicles from healthy individuals All authors de Menezes-Neto A, Sáez MJ, Lozano-Ramos I, Segui-Barber J, Martin-Jaular L, Ullate JM, Fernandez-Becerra C, Borrás FE, Del Portillo HA Journal J Extracell Vesicles Abstract Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major cha (show more...)Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of classical exosome markers. Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS. (hide) EV-METRIC 57% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Focus vesicles Vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography SEC Protein markers EV: CD81/ CD9/ GAPDH non-EV: Albumin Proteomics yes Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Characterization: Protein analysis Western Blot Detected EV-associated proteins GAPDH ELISA Detected EV-associated proteins GAPDH Characterization: Particle analysis NTA EM EM-type immune EM/ cryo EM Image type Close-up

	EV150003	1/2	Homo sapiens	Cell culture supernatant	dUC	Saari H	2015	56%
Study summary Full title Microvesicle- and exosome-mediated drug delivery enhances the cytotoxicity of Paclitaxel in autologous prostate cancer cells All authors Saari H, Lázaro-Ibáñez E, Viitala T, Vuorimaa-Laukkanen E, Siljander P, Yliperttula M Journal J Control Release Abstract BACKGROUND: Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate int (show more...)BACKGROUND: Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. METHODS: EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. RESULTS: Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. CONCLUSIONS: Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes / microvesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 52.47 (washing) Protein markers EV: CD63/ CD81/ CD9/ TSG101/ Alpha-tubulin non-EV: GAPDH Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Equal to or above 150,000 g Pelleting: time(min) 60 Wash: volume per pellet (ml) 1 Wash: Rotor Type TLA55 Wash: adjusted k-factor 52.47 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ CD9/ TSG101/ Alpha-tubulin Detected contaminants GAPDH ELISA Detected EV-associated proteins Alpha-tubulin Characterization: Particle analysis NTA EM EM-type transmission EM/ cryo EM Image type Close-up

	EV150016	2/2	Homo sapiens	Ascites	0.2 µm filter Density cushion dUC	Pospichalova V	2015	56%
Study summary Full title Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer All authors Pospichalova V, Svoboda J, Dave Z, Kotrbova A, Kaiser K, Klemova D, Ilkovics L, Hampl A, Crha I, Jandakova E, Minar L, Weinberger V, Bryja V Journal J Extracell Vesicles Abstract Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and quali (show more...)Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization. (hide) EV-METRIC 56% (97th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Ascites Focus vesicles exosomes / microvesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Density cushion + dUC Adj. k-factor 253.9 (pelleting) Protein markers EV: Alix/ CD63/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Ascites Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 190 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis Particle analysis: flow cytometry EM EM-type transmission EM/ immune EM Image type Close-up, Wide-field

	EV150002	1/1	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG (valid.)	Phinney DG	2015	56%
Study summary Full title Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs All authors Phinney DG, Di Giuseppe M, Njah J, Sala E, Shiva S, St Croix CM, Stolz DB, Watkins SC, Di YP, Leikauf GD, Kolls J, Riches DW, Deiuliis G, Kaminski N, Boregowda SV, McKenna DH, Ortiz LA Journal Nat Commun Abstract Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and (show more...)Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: CD63/ CD9/ MFGE8 non-EV: None Proteomics no EV density (g/ml) 1.09-1.14 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Bottom-up Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD9/ MFGE8 ELISA Detected EV-associated proteins MFGE8 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV150027	2/2	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC Sucrose-DG (valid.)	Kharaziha P	2015	56%
Study summary Full title Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel All authors Kharaziha P, Chioureas D, Rutishauser D, Baltatzis G, Lennartsson L, Fonseca P, Azimi A, Hultenby K, Zubarev R, Ullén A, Yachnin J, Nilsson S, Panaretakis T Journal Oncotarget Abstract Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) whi (show more...)Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) which remains a leading cause of cancer-related deaths, worldwide. The clinical usage of docetaxel has resulted in modest gains in survival, primarily due to the development of resistance. There are currently no clinical biomarkers available that predict whether a CRPC patient will respond or acquire resistance to this therapy. Comparative proteomics analysis of exosomes secreted from DU145 prostate cancer cells that are sensitive (DU145 Tax-Sen) or have acquired resistance (DU145 Tax-Res) to docetaxel, demonstrated significant differences in the amount of exosomes secreted and in their molecular composition. A panel of proteins was identified by proteomics to be differentially enriched in DU145 Tax-Res compared to DU145 Tax-Sen exosomes and was validated by western blotting. Importantly, we identified MDR-1, MDR-3, Endophilin-A2 and PABP4 that were enriched only in DU145 Tax-Res exosomes. We validated the presence of these proteins in the serum of a small cohort of patients. DU145 cells that have uptaken DU145 Tax-Res exosomes show properties of increased matrix degradation. In summary, exosomes derived from DU145 Tax-Res cells may be a valuable source of biomarkers for response to therapy. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ CD63/ CD81/ CD9/ TSG101/ CD82/ Rab5/ Caveolin1 non-EV: Cell organelle protein Proteomics yes EV density (g/ml) 1.12-1.19 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting: time(min) 120 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.2 Highest density fraction 2 Orientation Top-down Speed (g) 120000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ CD81/ CD9/ TSG101/ CD82/ Rab5/ Caveolin1 Detected contaminants Cell organelle protein ELISA Detected EV-associated proteins CD82/ Rab5/ Caveolin1 Characterization: Particle analysis NTA EM EM-type transmission EM Image type Close-up

	EV150010	1/1	Homo sapiens	Cell culture supernatant	dUC Iodixanol-DG	Keerthikumar S	2015	56%
Study summary Full title Proteogenomic analysis reveals exosomes are more oncogenic than ectosomes. All authors Keerthikumar S, Gangoda L, Liem M, Fonseka P, Atukorala I, Ozcitti C, Mechler A, Adda CG, Ang CS, Mathivanan S Journal Oncotarget Abstract Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocyti (show more...)Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through tumor-derived EVs. Follow up integrated bioinformatics analysis highlighted the enrichment of oncogenic cargo in exosomes and ectosomes. Interestingly, exosomes induced significant cell proliferation and migration in recipient cells compared to ectosomes confirming the oncogenic nature of exosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes. The integrative proteogenomics approach utilized in this study has the potential to identify disease biomarker candidates which can be later assayed in liquid biopsies obtained from cancer patients. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes / Ectosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG Adj. k-factor 276.6 (pelleting) Protein markers EV: Alix/ CD63/ CD81/ TSG101 non-EV: Cell organelle protein/ MMP2 Proteomics yes EV density (g/ml) 1.100 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 1060 Pelleting: rotor type SW40 Pelleting: adjusted k-factor 276.6 Density gradient Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Pelleting-wash: volume per pellet (mL) 1 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ CD81/ TSG101 Detected contaminants Cell organelle protein/ MMP2 Characterization: Particle analysis EM EM-type transmission EM/ atomic force EM Image type Wide-field

	EV150021	1/1	Homo sapiens	Cell culture supernatant	dUC Iodixanol-DG (valid.)	Duchez AC	2015	56%
Study summary Full title Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA All authors Duchez AC, Boudreau LH, Bollinger J, Belleannée C, Cloutier N, Laffont B, Mendoza-Villarroel RE, Lévesque T, Rollet-Labelle E, Rousseau M, Allaeys I, Tremblay JJ, Poubelle PE, Lambeau G, Pouliot M, Provost P, Soulet D, Gelb MH, Boilard E Journal Proc Natl Acad Sci U S A Abstract Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) cal (show more...)Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms. (hide) EV-METRIC 56% (93rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles microparticles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG (valid.) Protein markers EV: TSG101/ VDAC non-EV: None Proteomics no EV density (g/ml) 1.1-1.14 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 90 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Characterization: Protein analysis Western Blot Detected EV-associated proteins TSG101/ VDAC ELISA Detected EV-associated proteins VDAC Characterization: Particle analysis DLS

	EV150103	7/7	Homo sapiens	Cell culture supernatant	dUC	Dieudé M	2015	55%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 55% (91st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles apoptotic exosome-like vesicle Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: Syntenin/ Fibronectin/ TCTP/ proteasome-alpha3/ LG3 non-EV: GM130/ Tubulin Proteomics yes Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Apoptosis EV-producing cells HUVEC EV-harvesting Medium Serum free medium Cell viability 75 Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Protein Concentration Method microBCA Protein Concentration 300 Western Blot Detected EV-associated proteins Syntenin, Fibronectin, TCTP, proteasome-alpha3, LG3 Not detected contaminants GM130, Tubulin Proteomics database Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BDCantoII Special Order Research Product Hardware adjustment This high sensitivity Flow cytometer (hsFCM) is equipped with a small particle option. The forward scatter (FSC) on this dedicated equipment is coupled to a photomultiplier tube (PMT) with a 488 nm solid state;100mW output blue laser (rather than the conventional 20 mW);and includes a 633nmHeNe;20mW output red laser and a 405 nm solid state diode;50mW output violet laser. The hsFCM includes a FSC-PMT and a Fourier optical transformation unit;which reduces the background noise and increases the angle of diffusion;therby enhancing the detection of small-diameter particles. Calibration bead size 0.09,0.45,0.84,1,3.2 Report type Median Reported size (nm) 100-200 EV concentration Yes Particle yield 3.50E+07 particles/million cells EM EM-type Transmission-EM/ Immune-EM Image type Close-up, Wide-field

	EV150007	1/10	Homo sapiens	Cell culture supernatant	Commercial UF	Lobb RJ	2015	50%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 50% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Commercial + UF Protein markers EV: Flotilin1/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Commercial kit qEV Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV150007	4/10	Homo sapiens	Cell culture supernatant	0.2 µm filter UF	Lobb RJ	2015	50%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 50% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + UF Protein markers EV: CD63/ Flotilin1/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Flotilin1/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV150007	5/10	Homo sapiens	Cell culture supernatant	0.2 µm filter Iodixanol-DG UF	Lobb RJ	2015	50%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 50% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Iodixanol-DG + UF Protein markers EV: Flotilin1/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Density gradient Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Rotor type 50.2Ti Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis TRPS

	EV150007	7/10	Homo sapiens	Cell culture supernatant	0.2 µm filter Commercial	Lobb RJ	2015	50%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 50% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Commercial Protein markers EV: Flotilin1/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Filtration steps 0.22µm or 0.2µm Commercial kit Exo-spin Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Close-up

	EV150007	8/10	Homo sapiens	Cell culture supernatant	0.2 µm filter Commercial	Lobb RJ	2015	50%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 50% (90th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Commercial Protein markers EV: Flotilin1/ HSP70/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ HSP70/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Close-up

	EV150103	5/7	Homo sapiens	Cell culture supernatant	dUC	Dieudé M	2015	44%
Study summary Full title The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection. All authors Dieudé M, Bell C, Turgeon J, Beillevaire D, Pomerleau L, Yang B, Hamelin K, Qi S, Pallet N, Béland C, Dhahri W, Cailhier JF, Rousseau M, Duchez AC, Lévesque T, Lau A, Rondeau C, Gingras D, Muruve D, Rivard A, Cardinal H, Perreault C, Desjardins M, Boilard É, Thibault P, Hébert MJ Journal J Transl Med Abstract Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rej (show more...)Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles apoptotic body Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 127.9 (pelleting) Protein markers EV: Syntenin/ Fibronectin/ TCTP/ GM130/ Tubulin/ LG3 non-EV: None Proteomics yes Show all info Study aim Function, Biomarker, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Apoptosis EV-producing cells HUVEC EV-harvesting Medium Serum free medium Cell viability 75 Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 50,000 g and 100,000 g Pelleting: time(min) 1080 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 127.9 Characterization: Protein analysis Protein Concentration Method microBCA Protein Concentration 2000 Western Blot Detected EV-associated proteins Syntenin, Fibronectin, TCTP, GM130, Tubulin, LG3 Proteomics database Yes Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BDCantoII Special Order Research Product Hardware adjustment This high sensitivity Flow cytometer (hsFCM) is equipped with a small particle option. The forward scatter (FSC) on this dedicated equipment is coupled to a photomultiplier tube (PMT) with a 488 nm solid state;100mW output blue laser (rather than the conventional 20 mW);and includes a 633nmHeNe;20mW output red laser and a 405 nm solid state diode;50mW output violet laser. The hsFCM includes a FSC-PMT and a Fourier optical transformation unit;which reduces the background noise and increases the angle of diffusion;therby enhancing the detection of small-diameter particles. Calibration bead size 0.09,0.45,0.84,1,3.2 Report type Median Reported size (nm) 100-200 EV concentration Yes Particle yield 3.50E+07 particles/million cells EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV150031	1/1	Homo sapiens	Cell culture supernatant	dUC Iodixanol-DG (valid.)	van Niel G	2015	44%
Study summary Full title Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells All authors van Niel G, Bergam P, Di Cicco A, Hurbain I, Lo Cicero A, Dingli F, Palmulli R, Fort C, Potier MC, Schurgers LJ, Loew D, Levy D, Raposo G Journal Cell Rep Abstract Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related dise (show more...)Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV) within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles Endosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Iodixanol-DG (valid.) Protein markers EV: CD63/ TSG101/ ApoE non-EV: None Proteomics yes EV density (g/ml) 1.06-1.15 Show all info Study aim Biogenesis/Sorting Sample Species Homo sapiens Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 60 Density gradient Only used for validation of main results 1 Density medium Iodixanol Lowest density fraction 10 Highest density fraction 40 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ TSG101/ ApoE ELISA Detected EV-associated proteins ApoE Characterization: Particle analysis EM EM-type immune EM/ cryo EM Image type Close-up, Wide-field

	EV150051	1/3	Homo sapiens	Cell culture supernatant	dUC	Hoshino A	2015	44%
Study summary Full title Tumour exosome integrins determine organotropic metastasis All authors Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, Molina H, Kohsaka S, Di Giannatale A, Ceder S, Singh S, Williams C, Soplop N, Uryu K, Pharmer L, King T, Bojmar L, Davies AE, Ararso Y, Zhang T, Zhang H, Hernandez J, Weiss JM, Dumont-Cole VD, Kramer K, Wexler LH, Narendran A, Schwartz GK, Healey JH, Sandstrom P, Jørgen Labori K, Kure EH, Grandgenett PM, Hollingsworth MA, de Sousa M, Kaur S, Jain M, Mallya K, Batra SK, Jarnagin WR, Brady MS, Fodstad O, Muller V, Pantel K, Minn AJ, Bissell MJ, Garcia BA, Kang Y, Rajasekhar VK, Ghajar CM, Matei I, Peinado H, Bromberg J, Lyden D Journal Nature Abstract Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's gr (show more...)Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins ?6?4 and ?6?1 were associated with lung metastasis, while exosomal integrin ?v?5 was linked to liver metastasis. Targeting the integrins ?6?4 and ?v?5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: GAPDH non-EV: None Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: volume per pellet (ml) 20 Wash: Rotor Type 70Ti Wash: adjusted k-factor 156.9 Characterization: Protein analysis Western Blot Detected EV-associated proteins GAPDH ELISA Detected EV-associated proteins GAPDH Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV150012	1/2	Homo sapiens	Cell culture supernatant	dUC	Santi A	2015	44%
Study summary Full title Cancer associated fibroblasts transfer lipids and proteins to cancer cells through cargo vesicles supporting tumor growth All authors Santi A, Caselli A, Ranaldi F, Paoli P, Mugnaioni C, Michelucci E, Cirri P Journal Biochim Biophys Acta Mol Cell Res Abstract Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matri (show more...)Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matrix, form the structural scaffolding of organs. In solid tumors, interaction with cancer cells induces fibroblasts transdifferentiation into an activated form, which become a fundamental part of the tumor stroma. Within tumor microenvironment stromal and cancer cells engage a crosstalk that is mediated by soluble factors, cellcell contacts and extracellular vesicles trafficlking. Here we report that fibroblasts have the ability to transfer a remarkable amount of proteins and lipids to neighboring cells, in an ectosome-dependent fashion, identifying a novel and native property of these cells. Cancer-associated fibroblasts show an enhanced production and delivering of ectc:Jsomes to cancer cells compared to normal fibroblasts. As a consequence of this phenomenon, tumor cells increase their proliferation rate, indicating that ectosome-mediated trafficking could be a relevant mechanism mediating the trophic function of activated connective tissue on tumor cells. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles Vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: CD81/ Tubulin/ GAPDH non-EV: Cell organelle protein/ Alpha-enolase/ Vimentin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Tubulin/ GAPDH Detected contaminants Cell organelle protein/ Alpha-enolase/ Vimentin ELISA Detected EV-associated proteins Tubulin/ GAPDH Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV150012	2/2	Homo sapiens	Cell culture supernatant	dUC	Santi A	2015	44%
Study summary Full title Cancer associated fibroblasts transfer lipids and proteins to cancer cells through cargo vesicles supporting tumor growth All authors Santi A, Caselli A, Ranaldi F, Paoli P, Mugnaioni C, Michelucci E, Cirri P Journal Biochim Biophys Acta Mol Cell Res Abstract Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matri (show more...)Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matrix, form the structural scaffolding of organs. In solid tumors, interaction with cancer cells induces fibroblasts transdifferentiation into an activated form, which become a fundamental part of the tumor stroma. Within tumor microenvironment stromal and cancer cells engage a crosstalk that is mediated by soluble factors, cellcell contacts and extracellular vesicles trafficlking. Here we report that fibroblasts have the ability to transfer a remarkable amount of proteins and lipids to neighboring cells, in an ectosome-dependent fashion, identifying a novel and native property of these cells. Cancer-associated fibroblasts show an enhanced production and delivering of ectc:Jsomes to cancer cells compared to normal fibroblasts. As a consequence of this phenomenon, tumor cells increase their proliferation rate, indicating that ectosome-mediated trafficking could be a relevant mechanism mediating the trophic function of activated connective tissue on tumor cells. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles Vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: Integrin-beta1/ MMP2/ Tubulin/ GAPDH/ Vimentin/ Alpha-enolase non-EV: Cell organelle protein/ CD81 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 40 Characterization: Protein analysis Western Blot Detected EV-associated proteins Integrin-beta1/ MMP2/ Tubulin/ GAPDH/ Vimentin/ Alpha-enolase Detected contaminants Cell organelle protein/ CD81 ELISA Detected EV-associated proteins Integrin-beta1/ MMP2/ Tubulin/ GAPDH/ Vimentin/ Alpha-enolase Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV150003	2/2	Homo sapiens	Cell culture supernatant	dUC	Saari H	2015	44%
Study summary Full title Microvesicle- and exosome-mediated drug delivery enhances the cytotoxicity of Paclitaxel in autologous prostate cancer cells All authors Saari H, Lázaro-Ibáñez E, Viitala T, Vuorimaa-Laukkanen E, Siljander P, Yliperttula M Journal J Control Release Abstract BACKGROUND: Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate int (show more...)BACKGROUND: Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. METHODS: EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. RESULTS: Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. CONCLUSIONS: Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes / microvesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 52.47 (washing) Protein markers EV: CD63/ CD81/ CD9/ TSG101/ Alpha-tubulin/ GAPDH non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 60 Wash: volume per pellet (ml) 1 Wash: Rotor Type TLA55 Wash: adjusted k-factor 52.47 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ CD9/ TSG101/ Alpha-tubulin/ GAPDH ELISA Detected EV-associated proteins Alpha-tubulin/ GAPDH Characterization: Particle analysis NTA EM EM-type transmission EM/ cryo EM Image type Close-up

	EV150015	2/2	Homo sapiens	Cell culture supernatant	0.45 µm filter Density cushion dUC Sucrose-DG (valid.)	Paggetti J	2015	44%
Study summary Full title Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts All authors Paggetti J, Haderk F, Seiffert M, Janji B, Distler U, Ammerlaan W, Kim YJ, Adam J, Lichter P, Solary E, Berchem G, Moussay E Journal Blood Abstract Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metast (show more...)Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected ?-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.45 µm filter + Density cushion + dUC + Sucrose-DG (valid.) Protein markers EV: Alix/ CD63/ HSP90/ TSG101/ MHC2/ Rab5a/ HSP72/ Ago2/ AChE non-EV: None Proteomics yes EV density (g/ml) 1.15-1.17 Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.2 Highest density fraction 2.5 Orientation Top-down Speed (g) 110000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63/ HSP90/ TSG101/ MHC2/ Rab5a/ HSP72/ Ago2/ AChE ELISA Detected EV-associated proteins MHC2/ Rab5a/ HSP72/ Ago2/ AChE Characterization: Particle analysis Particle analysis: flow cytometry

	EV150006	1/2	Homo sapiens Mus musculus	Cell culture supernatant	0.2 µm filter dUC Sucrose-DG (valid.)	Melo SA	2015	44%
Study summary Full title Glypican-1 identifies cancer exosomes and detects early pancreatic cancer All authors Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, LeBleu VS, Mittendorf EA, Weitz J, Rahbari N, Reissfelder C, Pilarsky C, Fraga MF, Piwnica-Worms D, Kalluri R Journal Nature Abstract Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. (show more...)Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Protein markers EV: CD81/ Flotilin1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Wash: volume per pellet (ml) 35 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 150000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Flotilin1 Characterization: Particle analysis NTA EM EM-type transmission EM/ immune EM Image type Close-up

	EV150006	2/2	Homo sapiens	Serum	0.2 µm filter dUC Sucrose-DG (valid.)	Melo SA	2015	44%
Study summary Full title Glypican-1 identifies cancer exosomes and detects early pancreatic cancer All authors Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, LeBleu VS, Mittendorf EA, Weitz J, Rahbari N, Reissfelder C, Pilarsky C, Fraga MF, Piwnica-Worms D, Kalluri R Journal Nature Abstract Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. (show more...)Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy. (hide) EV-METRIC 44% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Sucrose-DG (valid.) Protein markers EV: Flotilin1 non-EV: None Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Serum Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting: time(min) 960 Wash: volume per pellet (ml) 11 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Orientation Bottom-up Speed (g) 150000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1 Characterization: Particle analysis NTA EM EM-type transmission EM/ immune EM Image type Close-up

	EV150007	3/10	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	Lobb RJ	2015	44%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Adj. k-factor 157.1 (pelleting) / 157.1 (washing) Protein markers EV: Flotilin1/ HSP70/ TSG101 non-EV: None Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 1 Wash: Rotor Type 50.2Ti Wash: adjusted k-factor 157.1 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ HSP70/ TSG101 Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV150011	1/1	Homo sapiens	Cell culture supernatant	dUC Sucrose-DG (valid.)	Lazar I	2015	44%
Study summary Full title Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines All authors Lazar I, Clement E, Ducoux-Petit M, Denat L, Soldan V, Dauvillier S, Balor S, Burlet-Schiltz O, Larue L, Muller C, Nieto L Journal Pigm Cell Melanoma R Abstract Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma (show more...)Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma-specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro-migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells' aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Sucrose-DG (valid.) Protein markers EV: CD81/ Flotilin1/ TSG101 non-EV: None Proteomics yes EV density (g/ml) 1.13-1.19 Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2.5 Orientation Top-down Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ Flotilin1/ TSG101 Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV150005	2/2	Mus musculus	Cell culture supernatant	0.2 µm filter dUC	Nojima H	2015	44%
Study summary Full title Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate All authors Nojima H, Freeman CM, Schuster RM, Japtok L, Kleuser B, Edwards MJ, Gulbins E, Lentsch AB Journal J Hepatol Abstract BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hep (show more...)BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. METHODS: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. RESULTS: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. CONCLUSIONS: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Protein markers EV: CD63/ CD81/ TSG101 non-EV: Cell organelle protein/ Beta-actin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ TSG101 Detected contaminants Cell organelle protein/ Beta-actin Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up, Wide-field

	EV150024	1/2	Homo sapiens	Cell culture supernatant	dUC	Hazan-Halevy I	2015	44%
Study summary Full title Cell-specific uptake of mantle cell lymphoma-derived exosomes by malignant and non-malignant B-lymphocytes All authors Hazan-Halevy I, Rosenblum D, Weinstein S, Bairey O, Raanani P, Peer D Journal Cancer Lett Abstract Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatm (show more...)Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatments are based on chemotherapeutics and new class of drugs (e.g. Ibrutinib(®)), which in most cases ends with tumor resistance and relapse. Therefore, further development of novel therapeutic modalities is needed. Exosomes are natural extracellular vesicles, which play an important role in intercellular communication. The specificity of exosome uptake by different target cells remains unknown. In this study, we observed that MCL exosomes are taken up rapidly and preferentially by MCL cells. Only a minor fraction of exosomes was internalized into T-cell leukemia and bone marrow stroma cell lines, when these cells were co-cultured with MCL cells. Moreover, MCL patients' exosomes were taken up by both healthy and patients' B-lymphocytes with no apparent internalization to T lymphocytes and NK cells. Exosome internalization was not inhibited by specific siRNA against caveolin1 and clathrin but was found to be mediated by a cholesterol-dependent pathway. These findings demonstrate natural specificity of exosomes to B-lymphocytes and ultimately might be used for therapeutic intervention in B cells malignancies. (hide) EV-METRIC 44% (85th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: CD63/ CD81/ TSG101/ CD19 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ TSG101/ CD19 Detected contaminants Calnexin ELISA Detected EV-associated proteins CD19 Flow cytometry specific beads Selected surface protein(s) Yes Characterization: Particle analysis NTA Particle analysis: flow cytometry EM EM-type transmission EM/ immune EM Image type Close-up

	EV150039	1/1	Homo sapiens	Semen	dUC SEC Sucrose-DG	Dubois L	2015	44%
Study summary Full title Proteomic profiling of detergent resistant membranes (lipid rafts) of prostasomes All authors Dubois L, Ronquist KK, Ek B, Ronquist G, Larsson A Journal Mol Cell Proteomics Abstract Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular (show more...)Prostasomes are exosomes derived from prostate epithelial cells through exocytosis by multivesicular bodies. Prostasomes have a bilayered membrane and readily interact with sperm. The membrane lipid composition is unusual with a high contribution of sphingomyelin at the expense of phosphatidylcholine and saturated and monounsaturated fatty acids are dominant. Lipid rafts are liquid-ordered domains that are more tightly packed than the surrounding nonraft phase of the bilayer. Lipid rafts are proposed to be highly dynamic, submicroscopic assemblies that float freely within the liquid disordered membrane bilayer and some proteins preferentially partition into the ordered raft domains. We asked the question whether lipid rafts do exist in prostasomes and, if so, which proteins might be associated with them. Prostasomes of density range 1.13-1.19g/ml were subjected to density gradient ultracentrifugation in sucrose fabricated by phosphate buffered saline (PBS) containing 1% Triton X-100 with capacity for banding at 1.10 g/ml, i.e. the classical density of lipid rafts. Prepared prostasomal lipid rafts (by gradient ultracentrifugation) were analyzed by mass spectrometry. The clearly visible band on top of 1.10g/ml sucrose in the Triton X-100 containing gradient was subjected to liquid chromatography-tandem MS and more than 370 lipid raft associated proteins were identified. Several of them were involved in intraluminal vesicle formation, e.g. tetraspanins, ESCRTs, and Ras-related proteins. This is the first comprehensive liquid chromatography-tandem MS profiling of proteins in lipid rafts derived from exosomes. Data are available via ProteomeXchange with identifier PXD002163. (hide) EV-METRIC 44% (83rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Semen Focus vesicles Prostasomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + SEC + Sucrose-DG Adj. k-factor 126 (pelleting) Protein markers EV: Flotilin1/ Flotillin2/ Clathrin non-EV: None Proteomics yes EV density (g/ml) 1.13-1.19 Show all info Study aim Omics Sample Species Homo sapiens Sample Type Semen Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Pelleting: rotor type 90Ti Pelleting: adjusted k-factor 126.0 Density gradient Density medium Sucrose Lowest density fraction 1 Highest density fraction 2 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ Flotillin2/ Clathrin ELISA Detected EV-associated proteins Flotillin2/ Clathrin Characterization: Particle analysis

	EV150001	1/1	Homo sapiens	Urine	SEC UF	Lozano-Ramos I	2015	43%
Study summary Full title Size-exclusion chromatography-based enrichment of extracellular vesicles from urine samples All authors Lozano-Ramos I, Bancu I, Oliveira-Tercero A, Armengol MP, Menezes-Neto A, Del Portillo HA, Lauzurica-Valdemoros R, Borràs FE Journal J Extracell Vesicles Abstract Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this inv (show more...)Renal biopsy is the gold-standard procedure to diagnose most of renal pathologies. However, this invasive method is of limited repeatability and often describes an irreversible renal damage. Urine is an easily accessible fluid and urinary extracellular vesicles (EVs) may be ideal to describe new biomarkers associated with renal pathologies. Several methods to enrich EVs have been described. Most of them contain a mixture of proteins, lipoproteins and cell debris that may be masking relevant biomarkers. Here, we evaluated size-exclusion chromatography (SEC) as a suitable method to isolate urinary EVs. Following a conventional centrifugation to eliminate cell debris and apoptotic bodies, urine samples were concentrated using ultrafiltration and loaded on a SEC column. Collected fractions were analysed by protein content and flow cytometry to determine the presence of tetraspanin markers (CD63 and CD9). The highest tetraspanin content was routinely detected in fractions well before the bulk of proteins eluted. These tetraspanin-peak fractions were analysed by cryo-electron microscopy (cryo-EM) and nanoparticle tracking analysis revealing the presence of EVs.When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, tetraspanin-peak fractions from urine concentrated samples contained multiple bands but the main urine proteins (such as Tamm-Horsfall protein) were absent. Furthermore, a preliminary proteomic study of these fractions revealed the presence of EV-related proteins, suggesting their enrichment in concentrated samples. In addition, RNA profiling also showed the presence of vesicular small RNA species.To summarize, our results demonstrated that concentrated urine followed by SEC is a suitable option to isolate EVs with low presence of soluble contaminants. This methodology could permit more accurate analyses of EV-related biomarkers when further characterized by -omics technologies compared with other approaches. (hide) EV-METRIC 43% (82nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles extracellular vesicles Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography SEC + UF Protein markers EV: CD63/ CD9 non-EV: Albumin/ Tamm-Horsfall glycoprotein Proteomics no TEM measurements 80-120 Show all info Study aim Technical Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Characterization: Particle analysis NTA EM EM-type cryo EM Image type Close-up, Wide-field

	EV150018	2/2	Mus musculus	Brain tissue	dUC Immunoaffinity (valid.) Sucrose-DG (valid.)	Asai H	2015	43%
Study summary Full title Depletion of microglia and inhibition of exosome synthesis halt tau propagation All authors Asai H, Ikezu S, Tsunoda S, Medalla M, Luebke J, Haydar T, Wolozin B, Butovsky O, Kügler S, Ikezu T Journal Nat Neurosci Abstract Accumulation of pathological tau protein is a major hallmark of Alzheimer's disease. Tau protein spr (show more...)Accumulation of pathological tau protein is a major hallmark of Alzheimer's disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus-based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target. (hide) EV-METRIC 43% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Brain tissue Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Immunoaffinity (valid.) + Sucrose-DG (valid.) Protein markers EV: AChE non-EV: None Proteomics no TEM measurements 106.4+-29.6 Show all info Study aim Function Sample Species Mus musculus Sample Type Brain tissue Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Wash: volume per pellet (ml) 60 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.25 Highest density fraction 2 Western Blot Detected EV-associated proteins AChE ELISA Detected EV-associated proteins AChE Characterization: Particle analysis EM EM-type immune EM Image type Close-up

	EV150007	2/10	Homo sapiens	Blood plasma	Commercial UF	Lobb RJ	2015	38%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 38% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Commercial + UF Protein markers EV: Flotilin1 non-EV: Cell organelle protein/ Albumin Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Commercial kit qEV Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1 Detected contaminants Cell organelle protein/ Albumin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV150007	9/10	Homo sapiens	Blood plasma	0.2 µm filter Commercial	Lobb RJ	2015	38%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 38% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Commercial Protein markers EV: Flotilin1 non-EV: Cell organelle protein/ Albumin Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1 Detected contaminants Cell organelle protein/ Albumin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Close-up

	EV150007	10/10	Homo sapiens	Blood plasma	0.2 µm filter Commercial	Lobb RJ	2015	38%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 38% (86th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Commercial Protein markers EV: Flotilin1 non-EV: Cell organelle protein/ Albumin Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Blood plasma Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Filtration steps 0.22µm or 0.2µm Commercial kit Exo-spin Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1 Detected contaminants Cell organelle protein/ Albumin Characterization: Particle analysis TRPS EM EM-type transmission EM Image type Wide-field

	EV150005	1/2	Mus musculus	Serum	Commercial	Nojima H	2015	38%
Study summary Full title Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate All authors Nojima H, Freeman CM, Schuster RM, Japtok L, Kleuser B, Edwards MJ, Gulbins E, Lentsch AB Journal J Hepatol Abstract BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hep (show more...)BACKGROUND & AIMS: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. METHODS: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. RESULTS: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. CONCLUSIONS: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (hide) EV-METRIC 38% (92nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Commercial Protein markers EV: CD63/ CD81/ TSG101 non-EV: Cell organelle protein/ Beta-actin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Serum Isolation Method Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ TSG101 Detected contaminants Cell organelle protein/ Beta-actin Characterization: Particle analysis DLS EM EM-type transmission EM Image type Close-up

	EV150004	2/2	Homo sapiens	Cell culture supernatant	0.2 µm filter Commercial UF	Clark DJ	2015	38%
Study summary Full title Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis All authors Clark DJ, Fondrie WE, Liao Z, Hanson PI, Fulton A, Mao L, Yang AJ Journal Anal Chem Abstract Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into t (show more...)Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as true exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression. (hide) EV-METRIC 38% (78th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Commercial + UF Protein markers EV: Alix/ CD63 non-EV: None Proteomics yes Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Filtration steps 0.22µm or 0.2µm Commercial kit qEV Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ CD63 Characterization: Particle analysis

	EV150013	1/1	Mus musculus	Cell culture supernatant	0.2 µm filter Commercial dUC	Zhu Y	2015	33%
Study summary Full title A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor-Associated Macrophages All authors Zhu Y, Chen X, Pan Q, Wang Y, Su S, Jiang C, Li Y, Xu N, Wu L, Lou X, Liu S Journal J Proteome Res Abstract Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extrac (show more...)Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment. (hide) EV-METRIC 33% (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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + Commercial + dUC Protein markers EV: Alix/ HSP90/ TSG101 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Omics Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 120 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ HSP90/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV150033	1/1	Mus musculus	Cell culture supernatant	dUC	DeClercq V	2015	33%
Study summary Full title Fatty acids increase adiponectin secretion through both classical and exosome pathways All authors DeClercq V, D'Eon B, McLeod RS Journal Biochim Biophys Act Cell Biol L Abstract Little is known about the effects of fatty acids on adiponectin oligomer assembly and trafficking. T (show more...)Little is known about the effects of fatty acids on adiponectin oligomer assembly and trafficking. The aim of this study was to examine the effects of different fatty acids on adiponectin transport and secretion in differentiated 3T3-L1 adipocytes. Subcellular fractionation and immunofluorescence microscopy revealed that the majority of cellular adiponectin was located in the endoplasmic reticulum (ER). Adiponectin secretion was increased by treatment with fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and several fatty acids changed the cellular localization of adiponectin. Adiponectin secretion has been shown to be altered by ER stress and interactions with ER chaperone proteins. However these mechanisms were not influenced by fatty acids, suggesting that alternative mechanisms must be responsible for the increased secretion of adiponectin observed with fatty acid treatment. Secretion of adiponectin was blocked by Brefeldin A, but we identified a minor pool of adiponectin that could be secreted from beyond the Brefeldin A block. Exosomes appeared to contribute to a minor amount of adiponectin secreted from the cell, and exosome release was increased by treatment with DHA. These data suggest that the ER is an important site of adiponectin accumulation and that treatment with long chain omega-3 fatty acids increases adiponectin release. Furthermore, the secretory pathway of adiponectin is complex, involving both the classical ER-Golgi pathway as well as unconventional secretory mechanisms such as an exosome-mediated pathway. (hide) EV-METRIC 33% (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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: Alix/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis

	EV150032	1/1	Bos bovis	Milk	0.2 µm filter dUC	Wolf T	2015	33%
Study summary Full title The Intestinal Transport of Bovine Milk Exosomes Is Mediated by Endocytosis in Human Colon Carcinoma Caco-2 Cells and Rat Small Intestinal IEC-6 Cells All authors Wolf T, Baier SR, Zempleni J Journal J Nutr Abstract BACKGROUND: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs i (show more...)BACKGROUND: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs in tissues and body fluids is encapsulated in exosomes, thereby conferring protection against degradation and a pathway for intestinal transport. MicroRNAs in cow milk are bioavailable in humans. OBJECTIVE: This research assessed the transport mechanism of bovine milk exosomes, and therefore microRNAs, in human and rodent intestinal cells. METHODS: The intestinal transport of bovine milk exosomes and microRNAs was assessed using fluorophore-labeled bovine milk exosomes in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. Transport kinetics and mechanisms were characterized using dose-response studies, inhibitors of vesicle transport, carbohydrate competitors, proteolysis of surface proteins on cells and exosomes, and transepithelial transport in transwell plates. RESULTS: Exosome transport exhibited saturation kinetics at 37°C [Michaelis constant (Km) = 55.5 ± 48.6 ?g exosomal protein/200 ?L of media; maximal transport rate = 0.083 ± 0.057 ng of exosomal protein · 81,750 cells(-1) · h(-1)] and decreased by 64% when transport was measured at 4°C, consistent with carrier-mediated transport in Caco-2 cells. Exosome uptake decreased by 61-85% under the following conditions compared with controls in Caco-2 cells: removal of exosome and cell surface proteins by proteinase K, inhibition of endocytosis and vesicle trafficking by synthetic inhibitors, and inhibition of glycoprotein binding by carbohydrate competitors. When milk exosomes, at a concentration of 5 times the Km, were added to the upper chamber in transwell plates, Caco-2 cells accumulated miR-29b and miR-200c in the lower chamber, and reverse transport was minor. Transport characteristics were similar in IEC-6 cells and Caco-2 cells, except that substrate affinity and transporter capacity were lower and higher, respectively. CONCLUSION: The uptake of bovine milk exosomes is mediated by endocytosis and depends on cell and exosome surface glycoproteins in human and rat intestinal cells. (hide) EV-METRIC 33% (62nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Adj. k-factor 176.2 (pelleting) Protein markers EV: CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Bos bovis Sample Type Milk Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type F37L-8x100 Pelleting: adjusted k-factor 176.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV150054	1/1	Mus musculus	Cell culture supernatant	0.2 µm filter dUC	Wang X	2015	33%
Study summary Full title Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis All authors Wang X, Gu H, Qin D, Yang L, Huang W, Essandoh K, Wang Y, Caldwell CC, Peng T, Zingarelli B, Fan GC Journal Sci Rep Abstract Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However (show more...)Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis. (hide) EV-METRIC 33% (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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Adj. k-factor 206.6 (pelleting) Protein markers EV: CD63/ CD81 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 45Ti Pelleting: adjusted k-factor 206.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81 Characterization: Particle analysis DLS EM

	EV150030	1/1	Homo sapiens	Urine	dUC	Perez-Hernandez J	2015	33%
Study summary Full title Increased Urinary Exosomal MicroRNAs in Patients with Systemic Lupus Erythematosus All authors Perez-Hernandez J, Forner MJ, Pinto C, Chaves FJ, Cortes R, Redon J Journal PLoS One Abstract There is increased interest in using microRNAs (miRNAs) as biomarkers in different diseases. Present (show more...)There is increased interest in using microRNAs (miRNAs) as biomarkers in different diseases. Present in body fluids, it is controversial whether or not they are mainly enclosed in exosomes, thus we studied if urinary miRNAs are concentrated inside exosomes and if the presence of systemic lupus erythematosus with or without lupus nephritis modifies their distribution pattern. We quantified specific miRNAs in urine of patients with systemic lupus erythematosus (n = 38) and healthy controls (n = 12) by quantitative reverse-transcription PCR in cell-free urine, exosome-depleted supernatant and exosome pellet obtained by ultracentrifugation. In control group, miR-335 (hide) EV-METRIC 33% (70th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 27.21 (pelleting) Protein markers EV: CD9/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 27.21 Wash: volume per pellet (ml) 2 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV150029	1/1	Homo sapiens	Urine	0.2 µm filter dUC UF	Øverbye A	2015	33%
Study summary Full title Identification of prostate cancer biomarkers in urinary exosomes All authors Øverbye A, Skotland T, Koehler CJ, Thiede B, Seierstad T, Berge V, Sandvig K, Llorente A Journal Oncotarget Abstract Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-spe (show more...)Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-specific molecules can be found in exosomes isolated from biological fluids. We have here investigated the proteome of urinary exosomes by using mass spectrometry to identify proteins differentially expressed in prostate cancer patients compared to healthy male controls. In total, 15 control and 16 prostate cancer samples of urinary exosomes were analyzed. Importantly, 246 proteins were differentially expressed in the two groups. The majority of these proteins (221) were up-regulated in exosomes from prostate cancer patients. These proteins were analyzed according to specific criteria to create a focus list that contained 37 proteins. At 100% specificity, 17 of these proteins displayed individual sensitivities above 60%. Even though several of these proteins showed high sensitivity and specificity for prostate cancer as individual biomarkers, combining them in a multi-panel test has the potential for full differentiation of prostate cancer from non-disease controls. The highest sensitivity, 94%, was observed for transmembrane protein 256 (TM256; chromosome 17 open reading frame 61). LAMTOR proteins were also distinctly enriched with very high specificity for patient samples. TM256 and LAMTOR1 could be used to augment the sensitivity to 100%. Other prominent proteins were V-type proton ATPase 16 kDa proteolipid subunit (VATL), adipogenesis regulatory factor (ADIRF), and several Rab-class members and proteasomal proteins. In conclusion, this study clearly shows the potential of using urinary exosomes in the diagnosis and clinical management of prostate cancer. (hide) EV-METRIC 33% (70th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + UF Adj. k-factor 156.9 (pelleting) / 276.6 (washing) Protein markers EV: CD81/ CD9/ TSG101 non-EV: Tamm-Horsfall glycoprotein Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Isolation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: Rotor Type SW40 Wash: adjusted k-factor 276.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD81/ CD9/ TSG101 Detected contaminants Tamm-Horsfall glycoprotein Characterization: Particle analysis EM EM-type immune EM Image type Wide-field

	EV150007	6/10	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC Iodixanol-DG	Lobb RJ	2015	33%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 33% (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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC + Iodixanol-DG Adj. k-factor 157.1 (pelleting) / 157.1 (washing) Protein markers EV: Flotilin1/ TSG101 non-EV: None Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting: time(min) 90 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 1 Wash: Rotor Type 50.2Ti Wash: adjusted k-factor 157.1 Density gradient Density medium Iodixanol Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Rotor type 50.2Ti Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Flotilin1/ TSG101 Characterization: Particle analysis TRPS

	EV150028	1/1	Homo sapiens	Cell culture supernatant	0.2 µm filter dUC	Koch R	2015	33%
Study summary Full title Nuclear trapping through inhibition of exosomal export by indomethacin increases cytostatic efficacy of doxorubicin and pixantrone All authors Koch R, Aung T, Vogel D, Chapuy B, Wenzel D, Becker S, Sinzig U, Venkataramani V, von Mach T, Jacob R, Truemper L, Wulf GG Journal Clin Cancer Res Abstract PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with agg (show more...)PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with aggressive B-cell lymphoma, tumor cell susceptibility to chemotherapy varies, with mostly fatal outcome in cases of resistant disease. We and others have shown before that export of cytostatic drugs contributes to drug resistance. Now we provide a novel approach to overcome exosome-mediated drug resistance in aggressive B-cell lymphomas. EXPERIMENTAL DESIGN: We used well-established centrifugation protocols to purify exosomes from DLBCL cell lines and detected anthracyclines using FACS and HPLC. We used shRNA knockdown of ABCA3 to determine ABCA3 dependence of chemotherapy susceptibility and monitored ABCA3 expression after indomethacin treatment using qPCR. Finally, we established an in vivo assay using a chorioallantoic membrane (CAM) assay to determine the synergy of anthracycline and indomethacin treatment. RESULTS: We show increased efficacy of the anthracycline doxorubicin and the anthracenedione pixantrone by suppression of exosomal drug resistance with indomethacin. B-cell lymphoma cells in vitro efficiently extruded doxorubicin and pixantrone, in part compacted in exosomes. Exosomal biogenesis was critically dependent on the expression of the ATP-transporter A3 (ABCA3). Genetic or chemical depletion of ABCA3 augmented intracellular retention of both drugs and shifted the subcellular drug accumulation to prolonged nuclear retention. Indomethacin increased the cytostatic efficacy of both drugs against DLBCL cell lines in vitro and in vivo in a CAM assay. CONCLUSIONS: We propose pretreatment with indomethacin toward enhanced antitumor efficacy of anthracyclines and anthracenediones. Clin Cancer Res; 1-10. ©2015 AACR. (hide) EV-METRIC 33% (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 electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Isolation method: density gradient, at least as validation of results attributed to EVs EV density Isolation method: reporting of obtained EV density ultracentrifugation specifics Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Focus vesicles exosomes Isolation protocol Isolation protocol Gives a short, non-chronological overview of the different steps of the isolation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography 0.2 µm filter + dUC Adj. k-factor 130.7 (pelleting) Protein markers EV: CD63/ CD81/ CD9/ "ADAM10/ Flotillin2/ GAPDH" non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Isolation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 130.7 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ CD81/ CD9/ "ADAM10/ Flotillin2/ GAPDH" ELISA Detected EV-associated proteins "ADAM10/ Flotillin2/ GAPDH" Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV150026	1/1	Caenorhabditis elegans	Cell culture supernatant	dUC UF	Hyenne V	2015	33%
Study summary Full title RAL-1 controls multivesicular body biogenesis and exosome secretion All authors Hyenne V, Apaydin A, Rodriguez D, Spiegelhalter C, Hoff-Yoessle S, Diem M, Tak S, Lefebvre O, Schwab Y, Goetz JG, Labouesse M Journal J Cell Biol Abstract Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plas (show more...)Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1-deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release. (hide) EV-METRIC 33% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable

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