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Results list Most used separation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV160001	1/1	Mus musculus	Cell culture supernatant	DG dUC	Stremersch S	2016	87%
Study summary Full title Comparing exosome-like vesicles with liposomes for the functional cellular delivery of small RNAs. All authors Stremersch S, Vandenbroucke RE, Van Wonterghem E, Hendrix A, De Smedt SC, Raemdonck K Journal J Control Release Abstract Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natu (show more...)Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natural carriers for biomolecule transfer between cells. This unique feature rationalizes their exploitation as bio-inspired drug delivery systems. However, the therapeutic application of ELVs is hampered by the lack of efficient and reproducible drug loading methods, in particular for therapeutic macromolecules. To overcome this limitation, we present a generic method to attach siRNA to the surface of isolated ELVs by means of a cholesterol anchor. Despite a feasible uptake in both a dendritic and lung epithelial cell line, B16F10- and JAWSII-derived ELVs were unable to functionally deliver the associated small RNAs, neither exogenous cholesterol-conjugated siRNA nor endogenous miRNA derived from the melanoma producer cell. The latter results were confirmed both for purified ELVs and ELVs delivered via a transwell co-culture set-up. In contrast, simple anionic fusogenic liposomes were able to induce a marked siRNA-mediated gene knockdown under equal experimental conditions, both indicating successful cytosolic delivery of surface-bound cholesterol-conjugated siRNA and further underscoring the incapacity of the here evaluated ELVs to guide cytosolic delivery of small RNAs. In conclusion, we demonstrate that a more in-depth understanding of the biomolecular delivery mechanism and specificity is required before ELVs can be envisioned as a generic siRNA carrier. (hide) EV-METRIC 87% (98th 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome-like vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Protein markers EV: CD81/ HSP70/ beta-actin/ CD63 non-EV: GM130/ Calreticulin Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells B16F10, JAWSII EV-harvesting Medium EV-depleted serum Preparation of EDS Ultrafiltration (MWCO 300 kDa) Cell viability 95 Separation Method Differential ultracentrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Density gradient Only used for validation of main results Yes Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 0.125 Highest density fraction 0.5 Sample volume (mL) 1 Orientation Top-down (sample migrates downwards) Rotor type SW 55 Ti Speed (g) 200000 Duration (min) 900 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 5 Pelleting: duration (min) 150 Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Pelleting: adjusted k-factor 115.5 Pelleting-wash: volume per pellet (mL) 5 Pelleting-wash: duration (min) 70 Pelleting-wash: rotor type 115.5 Pelleting-wash: speed (g) SW 55 Ti Pelleting-wash: adjusted k-factor 115.5 EV-subtype Used subtypes NO Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63, CD81, HSP70, beta-actin Flow cytometry specific beads Selected surface protein(s) CD63 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-300 EV concentration Yes Particle yield 1000 EM EM-type Cryo-EM Image type Close-up

	EV160004	2/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	66%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 66% (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 71.08 (pelleting) Protein markers EV: Annexin-A1/ CD90/Thy1.1 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation 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 MLS-50 Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 71.08 Density gradient Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0M Highest density fraction 1.4M Sample volume (mL) 1.5 Orientation Bottom-up (sample migrates upwards) Rotor type MLS-50 Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 60 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 138.3 Characterization: Protein analysis Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Annexin-A1, CD90/Thy1.1 Characterization: Particle analysis EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 20-200 Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.

	EV160004	5/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	66%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 66% (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 1421 (pelleting) Protein markers EV: Annexin-A1/ CD90/Thy1.1 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 120 Pelleting: rotor type MLS-50 Pelleting: speed (g) 10000 Pelleting: adjusted k-factor 1421. Density gradient Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 3 Lowest density fraction 0M Highest density fraction 1.4M Sample volume (mL) 1.5 Orientation Bottom-up (sample migrates upwards) Rotor type MLS-50 Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 60 Pelleting: rotor type SW 40 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 138.3 Characterization: Protein analysis Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Annexin-A1, CD90/Thy1.1 Characterization: Particle analysis EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 20-200 Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation.

	EV200044	1/1	Homo sapiens	Blood plasma	Precipitation Density gradient	Zhang YN	2016	63%
Study summary Full title Extracellular Vesicle Proteins Associated with Systemic Vascular Events Correlate with Heart Failure: An Observational Study in a Dyspnoea Cohort. All authors Zhang YN, Vernooij F, Ibrahim I, Ooi S, Gijsberts CM, Schoneveld AH, Sen KW, den Ruijter HM, Timmers L, Richards AM, Jong CT, Mazlan I, Wang JW, Lam CS, de Kleijn DP Journal PLoS One Abstract BACKGROUND: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma (show more...)BACKGROUND: SerpinF2, SerpinG1, CystatinC and CD14 are involved in inflammatory processes and plasma extracellular vesicle (EV) -levels of these proteins have been reported to be associated with systemic vascular events. Evidence is accumulating that inflammatory processes may play a pivotal role both in systemic vascular events and in heart failure. Therefore, we studied the association between plasma extracellular vesicle SerpinF2-, SerpinG1-, CystatinC and CD14-levels and the occurrence of acute heart failure in patients. METHODS AND RESULT: Extracellular vesicle protein levels of SerpinG1, SerpinF2, CystatinC and CD14 were measured in an observational study of 404 subjects presenting with dysponea at the emergency department (4B-cohort). Plasma extracellular vesicles were precipitated in a total extracellular vesicles (TEX)-fraction and in separate LDL- and HDL-subfractions. Extracellular vesicle protein levels were measured with a quantitative immune assay in all 3 precipitates. Out of 404 subjects, 141 (35%) were diagnosed with acutely decompensated heart failure. After correction for confounders (including comorbidities and medications), levels of CD14 in the HDL-fraction (OR 1.53, p = 0.01), SerpinF2 in the TEX-and LDL-fraction (ORs respectively 0.71 and 0.65, p<0.05) and SerpinG1 in the TEX-fraction (OR 1.55, p = 0.004) were statistically significantly related to heart failure. Furthermore, extracellular vesicle CD14- and SerpinF2-levels were significantly higher in heart failure patients with preserved ejection fraction than in those with reduced ejection fraction. CONCLUSION: Extracellular vesicle levels of CD14, SerpinG1 and SerpinF2 are associated with the occurrence of heart failure in subjects suspected for acute heart failure, suggesting common underlying pathophysiological mechanisms for heart failure and vascular events. (hide) EV-METRIC 63% (95th 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Healthy test subject Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography Precipitation + Density gradient Protein markers EV: CD14/ SerpinC1/ SerpinG1/ CystC/ SerpinF2 non-EV: None Proteomics no EV density (g/ml) 1.02-1.17 Show all info Study aim Biomarker/New methodological development Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Healthy test subject Separation Method Density gradient Only used for validation of main results Yes Density medium Iodixanol Type Discontinuous Number of initial discontinuous layers 5 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 10.5 Sample volume (mL) 0.5 Orientation Top-down Rotor type SW 41 Ti Speed (g) 200000 Duration (min) 1080 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 8 Pelleting: duration (min) 60 Pelleting: rotor type SW 41 Ti Pelleting: speed (g) 200000 Characterization: Protein analysis Western Blot Detected EV-associated proteins CD9 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD14/ SerpinC1/ SerpinG1/ CystC/ SerpinF2 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-120

	EV160008	1/3	Rattus norvegicus	Cell culture supernatant	dUC	Cianciaruso C	2016	55%
Study summary Full title Primary Human and Rat β-Cells Release the Intracellular Autoantigens GAD65, IA-2, and Proinsulin in Exosomes Together With Cytokine-Induced Enhancers of Immunity. All authors Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S Journal Diabetes Abstract The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide) EV-METRIC 55% (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: GAD67/ GAD65/ Insulin/ PDI/ Flotillin-1/ CD9/ IA-2 non-EV: Gp96/ Calreticulin Proteomics yes Show all info Study aim Function, Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Primary pancreatic islets EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 87.5 Separation 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 Pelleting: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, Flotillin-1, GAD65, GAD67, IA-2, PDI Not detected contaminants Calreticulin, Gp96 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Proteomics database No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 139 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 120

	EV160005	1/2	Homo sapiens	Bronchoalveolar lavage fluid	dUC Filtration	Héliot A	2016	55%
Study summary Full title Smoker extracellular vesicles influence status of human bronchial epithelial cells. All authors Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ Journal Int J Hyg Environ Health Abstract Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide) EV-METRIC 55% (33rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bronchoalveolar lavage fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 126 (pelleting) / 126 (washing) Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Sample Condition Control condition Separation 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 SW 55 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 126.0 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 126.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Characterization: Particle analysis NTA Report type Size range/distribution;Mean Reported size (nm) 138.1±2.2 EV concentration Yes Particle yield 2.48E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Extra information UC rotors added to the EV-TRACK entry after publication

	EV160005	2/2	Homo sapiens	Bronchoalveolar lavage fluid	dUC Filtration	Héliot A	2016	55%
Study summary Full title Smoker extracellular vesicles influence status of human bronchial epithelial cells. All authors Héliot A, Landkocz Y, Roy Saint-Georges F, Gosset P, Billet S, Shirali P, Courcot D, Martin PJ Journal Int J Hyg Environ Health Abstract Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for (show more...)Cigarette smoking is a habit that has spread all over the world and is a significant risk factor for many diseases including cardiovascular disease, chronic obstructive pulmonary disease (COPD), asthma and lung cancer. Evaluation and understanding of tobacco health effects are of major interest worldwide and answer to important societal concerns. Identification of new biomarkers of exposure to tobacco smoke potentially implicated in COPD or lung carcinogenesis would allow a better observation of tobacco exposed population, thanks to screening establishment at reversible stages of pathological processes. In this study, we questioned whether cigarette smoking alters miRNA profiles of Extracellular Vesicles (EVs) present in human Broncho Alveolar Lavages (BALs), which could affect surrounding normal bronchial epithelial cells status. To this aim, BALs were carried out on 10 Smokers and 10 Non-Smokers, and EVs were isolated from the supernatants and characterized. We then compared the amount of 10 microRNAs (miRNAs) present in Smokers versus Non-Smokers BAL EVs and performed statistical analysis to discuss the biological significance by the smoking status and to evaluate BAL EV miRNAs as potential biomarkers of tobacco exposure. Finally, we tested the effects of smokers versus non-smokers EVs on human bronchial epithelial cells (BEAS-2B) to compare their influence on the cells status. Our study shows for the first time in human samples that smoking can alter lung EV profile that can influence surrounding bronchial epithelial cells. (hide) EV-METRIC 55% (33rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Bronchoalveolar lavage fluid Sample origin Smokers Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 126 (pelleting) / 126 (washing) Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker Sample Species Homo sapiens Sample Type Bronchoalveolar lavage fluid Sample Condition Smokers Separation 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 SW 55 Ti Pelleting: speed (g) 110000 Pelleting: adjusted k-factor 126.0 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 110000 Wash: adjusted k-factor 126.0 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD63, CD81 Characterization: Particle analysis NTA Report type Size range/distribution;Mean Reported size (nm) 139.8±3.3 EV concentration Yes Particle yield 1.94E+11 particles/ml start sample EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 30-200 Extra information UC rotors added to the EV-TRACK entry after publication

	EV160004	1/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (43rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 83.68 (pelleting) Protein markers EV: CD44/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation 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) 65 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 200000 Pelleting: adjusted k-factor 83.68 Density gradient Density medium Iodixanol Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV160004	3/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (43rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 167.3 (pelleting) Protein markers EV: CD44/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation 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) 65 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 167.3 Density gradient Density medium Iodixanol Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV160004	4/5	Equus caballus	Synovial fluid	DG dUC	Boere J	2016	55%
Study summary Full title Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles. All authors Boere J, van de Lest CH, Libregts SF, Arkesteijn GJ, Geerts WJ, Nolte-'t Hoen EN, Malda J, van Weeren PR, Wauben MH Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important f (show more...)Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. (hide) EV-METRIC 55% (43rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Synovial fluid Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Adj. k-factor 1673 (pelleting) Protein markers EV: CD44/ CD9 non-EV: None Proteomics no Show all info Study aim Biomarker, New methodological development Sample Species Equus caballus Sample Type Synovial fluid Sample Condition Control condition Separation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting: time(min) 35 Pelleting: rotor type SW 60 Ti Pelleting: speed (g) 10000 Pelleting: adjusted k-factor 1673. Density gradient Density medium Iodixanol Type Continuous Number of initial discontinuous layers 15 Highest density fraction 0.51 Sample volume (mL) 1.75 Orientation Bottom-up (sample migrates upwards) Rotor type SW 40 Ti Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 19 Pelleting: duration (min) 60 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, CD44 Flow cytometry Type of Flow cytometry BD-Influx Hardware adjustments optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1,0.2 Characterization: Particle analysis Particle analysis: flow cytometry Flow cytometer type BD-Influx Hardware adjustment optimized jet-in-air-based BD Influx flow cytometer Calibration bead size 0.1;0.2 EV concentration Yes Particle yield 7.00E+08 particles/ml start sample Extra information Importantly, synovial fluid samples were pre-treated with hyaluronidase prior to ultracentrifugation. Concentration calculated as sum of EVs recovered after all pelleting steps (10K, 100K, 200K).

	EV160007	1/3	Homo sapiens	Blood plasma	dUC Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Acute myeloid leukemia Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration + SEC Protein markers EV: TSG101/ PD-1/ CD123/ CD96/ CD44/ CD34/ Pro-TGFbeta1 LAP/ Pro-TGFbeta1LAP/ PD-L1/ CLL-1/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Acute myeloid leukemia Separation 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 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 66 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101, CD44, CD34, CD123, CD96, CLL-1, Pro-TGFbeta1 LAP, PD-1, PD-L1 Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 77-92 EV concentration Yes Particle yield 8.90E+10 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV160007	2/3	Homo sapiens	Blood plasma	dUC Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Head and neck squamous cell carcinoma Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration + SEC Protein markers EV: TSG101/ CD39/ PD-1/ CD73/ Cox2/ Fas/ FasL/ PD-L1/ HSP70/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Head and neck squamous cell carcinoma Separation 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 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 123 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-1, PD-L1 Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 73-76 EV concentration Yes Particle yield 1.10E+11 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV160007	3/3	Homo sapiens	Blood plasma	dUC Filtration SEC	Hong CS	2016	50%
Study summary Full title Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. All authors Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL Journal J Extracell Vesicles Abstract OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integri (show more...)OBJECTIVE: Isolation from human plasma of exosomes that retain functional and morphological integrity for probing their protein, lipid and nucleic acid content is a priority for the future use of exosomes as biomarkers. A method that meets these criteria and can be scaled up for patient monitoring is thus desirable. METHODS: Plasma specimens (1 mL) of patients with acute myeloid leukaemia (AML) or a head and neck squamous cell carcinoma (HNSCC) were differentially centrifuged, ultrafiltered and fractionated by size exclusion chromatography in small disposable columns (mini-SEC). Exosomes were eluted in phosphate-buffered saline and were evaluated by qNano for particle size and counts, morphology by transmission electron microscopy, protein content, molecular profiles by western blots, and for ability to modify functions of immune cells. RESULTS: Exosomes eluting in fractions #3-5 had a diameter ranging from 50 to 200 nm by qNano, with the fraction #4 containing the bulk of clean, unaggregated exosomes. The exosome elution profiles remained constant for repeated runs of the same plasma. Larger plasma volumes could be fractionated running multiple mini-SEC columns in parallel. Particle concentrations per millilitre of plasma in #4 fractions of AML and HNSCC were comparable and were higher (p<0.003) than those in normal controls. Isolated AML exosomes co-incubated with normal human NK cells inhibited NKG2D expression levels (p<0.004), and HNSCC exosomes suppressed activation (p<0.01) and proliferation of activated T lymphocytes (p<0.03). CONCLUSIONS: Mini-SEC allows for simple and reproducible isolation from human plasma of exosomes retaining structural integrity and functional activity. It enables molecular/functional analysis of the exosome content in serial specimens of human plasma for clinical applications. (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration + SEC Protein markers EV: TSG101/ CD39/ CD123/ PD-1/ CD73/ Cox2/ Fas/ Pro-TGFbeta1 LAP/ Pro-TGFbeta1LAP/ FasL/ PD-L1/ CLL-1/ HSP70/ CD9 non-EV: None Proteomics no Show all info Study aim Function, Biomarker, New methodological development, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Blood plasma Sample Condition Control condition Separation 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 Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 1 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method BCA Protein Concentration 32 Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, TSG101, CD123, CLL-1, Pro-TGFbeta1 LAP, PD-1, HSP70, Cox2, CD73, CD39, Fas, FasL, PD-L1 Characterization: Particle analysis TRPS Report type Mean Reported size (nm) 69-76 EV concentration Yes Particle yield 7.00E+09 particles/ml start sample EM EM-type Transmission-EM Image type Wide-field

	EV160000	1/1	Homo sapiens	Milk	DG dUC	van Herwijnen MJ	2016	50%
Study summary Full title Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components. All authors van Herwijnen MJ, Zonneveld MI, Goerdayal S, Nolte-'t Hoen EN, Garssen J, Stahl B, Maarten Altelaar AF, Redegeld FA, Wauben MH Journal Mol Cell Proteomics Abstract Breast milk contains several macromolecular components with distinctive functions, whereby milk fat (show more...)Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome. (hide) EV-METRIC 50% (89th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + dUC Protein markers EV: Flotillin-1/ CD9 non-EV: None Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Milk Sample Condition Control condition Separation Method Differential ultracentrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Density gradient Density medium Sucrose Type Continuous Lowest density fraction 0.4M Highest density fraction 2.5M Sample volume (mL) 6.5 Orientation Top-down (sample migrates downwards) Rotor type SW 40 Ti Speed (g) 192000 Duration (min) 900-1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: volume per fraction 38.5 Pelleting: duration (min) 65 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Characterization: Protein analysis PMID previous EV protein analysis Proteomics;Western blot Western Blot Lysis buffer provided? Yes Detected EV-associated proteins CD9, Flotillin-1 Proteomics database Yes PMID previous EV particle analysis 25206958 Extra information A different gradient protocol was used in the publication of the same group that was referred to for additional protein and particle analysis of milk-derived extracellular vesicles (PMID: 25206958).

	EV160009	1/2	Homo sapiens	Cell culture supernatant	dUC Filtration	Benedikter BJ	2016	44%
Study summary Full title Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants. All authors Benedikter BJ, Volgers C, van Eijck PH, Wouters EFM, Savelkoul PHM, Reynaert NL, Haenen GRMM, Rohde GGU, Weseler AR, Stassen FRM Journal J Cell Sci Abstract INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) wi (show more...)INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. (hide) EV-METRIC 44% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Exposed to cigarette smoke extract Focus vesicles extracellular vesicle / exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 133.2 (pelleting) Protein markers EV: CD63 non-EV: grp94 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Exposed to cigarette smoke extract EV-producing cells BEAS2B EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability 75 Separation 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) 150 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 117734 Pelleting: adjusted k-factor 133.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected contaminants grp94 Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 60-300 EV concentration Yes EM EM-type Cryo-EM Image type Close-up Report size (nm) 50-160

	EV160009	2/2	Homo sapiens	Cell culture supernatant	dUC Filtration	Benedikter BJ	2016	44%
Study summary Full title Cigarette smoke extract induced exosome release is mediated by depletion of exofacial thiols and can be inhibited by thiol-antioxidants. All authors Benedikter BJ, Volgers C, van Eijck PH, Wouters EFM, Savelkoul PHM, Reynaert NL, Haenen GRMM, Rohde GGU, Weseler AR, Stassen FRM Journal J Cell Sci Abstract INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) wi (show more...)INTRODUCTION: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH). METHODS: BEAS-2B cells were exposed for 24h to CSE, H2O2, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH. EVs in media were measured using CD63+CD81+ bead-coupled flow cytometry or tunable resistive pulse sensing (TRPS). For characterization by Western Blotting, cryo-transmission electron microscopy and TRPS, EVs were isolated using ultracentrifugation. Glutathione disulfide and GSH in cells were assessed by a GSH reductase cycling assay, and exofacial thiols using Flow cytometry. RESULTS: CSE augmented the release of the EV subtype exosomes, which could be prevented by scavenging thiol-reactive components using NAC or GSH. Among thiol-reactive CSE components, H2O2 had no effect on exosome release, whereas acrolein imitated the NAC-reversible exosome induction. The exosome induction by CSE and acrolein was paralleled by depletion of cell surface thiols. Membrane impermeable thiol blocking agents, but not specific inhibitors of the exofacially located thiol-dependent enzyme PDI, stimulated exosome release. SUMMARY/CONCLUSION: Thiol-reactive compounds like acrolein account for CSE-induced exosome release by reacting with cell surface thiols. As acrolein is produced endogenously during inflammation, it may influence exosome release not only in smokers, but also in ex-smokers with chronic obstructive pulmonary disease. NAC and GSH prevent acrolein- and CSE-induced exosome release, which may contribute to the clinical benefits of NAC treatment. (hide) EV-METRIC 44% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle / exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Adj. k-factor 133.2 (pelleting) Protein markers EV: CD63 non-EV: grp94 Proteomics no Show all info Study aim Biogenesis/cargo sorting Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells BEAS2B EV-harvesting Medium EV-depleted serum Preparation of EDS >=18h at >= 100,000g Cell viability 100 Separation 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) 150 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 117734 Pelleting: adjusted k-factor 133.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Not detected contaminants grp94 Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 60-300 EV concentration Yes EM EM-type Cryo-EM Image type Close-up Report size (nm) 50-160

	EV160008	2/3	Homo sapiens	Cell culture supernatant	dUC	Cianciaruso C	2016	44%
Study summary Full title Primary Human and Rat β-Cells Release the Intracellular Autoantigens GAD65, IA-2, and Proinsulin in Exosomes Together With Cytokine-Induced Enhancers of Immunity. All authors Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S Journal Diabetes Abstract The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide) EV-METRIC 44% (79th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: Insulin/ GAD65/ Flotillin-1/ CD9/ IA-2 non-EV: None Proteomics yes Show all info Study aim Function, Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Primary human pancreatic islets, primary rat pancreatic islets, INS1 cell line EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 87.5 Separation 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 Pelleting: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9, Flotillin-1, GAD65, IA-2 ELISA Antibody details provided? Yes Lysis buffer provided? Yes Proteomics database No Characterization: Particle analysis NTA Report type Mode Reported size (nm) 143 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 120

	EV160017	1/8	Homo sapiens	Cell culture supernatant	ExoQuick	Li L	2016	37%
Study summary Full title Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. All authors Li L, Li C, Wang S, Wang Z, Jiang J, Wang W, Li X, Chen J, Liu K, Li C, Zhu G. Journal Cancer Res Abstract Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient o (show more...)Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. (hide) EV-METRIC 37% (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography ExoQuick Protein markers EV: CD81/ CD63 non-EV: Tubulin Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Cal-27 Oral squamous cell carcinoma EV-harvesting Medium Not specified Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Tubulin EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 50-200

	EV160017	2/8	Homo sapiens	Cell culture supernatant	ExoQuick	Li L	2016	37%
Study summary Full title Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. All authors Li L, Li C, Wang S, Wang Z, Jiang J, Wang W, Li X, Chen J, Liu K, Li C, Zhu G. Journal Cancer Res Abstract Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient o (show more...)Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. (hide) EV-METRIC 37% (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Hypoxic condition (1% O2) Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography ExoQuick Protein markers EV: CD81/ CD63 non-EV: Tubulin Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Hypoxic condition (1% O2) EV-producing cells Cal-27 Oral squamous cell carcinoma EV-harvesting Medium Not specified Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Tubulin EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 50-200

	EV160017	4/8	Homo sapiens	Cell culture supernatant	ExoQuick	Li L	2016	37%
Study summary Full title Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. All authors Li L, Li C, Wang S, Wang Z, Jiang J, Wang W, Li X, Chen J, Liu K, Li C, Zhu G. Journal Cancer Res Abstract Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient o (show more...)Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. (hide) EV-METRIC 37% (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography ExoQuick Protein markers EV: CD81/ CD63 non-EV: Tubulin Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells SCC-9 Oral squamous cell carcinoma EV-harvesting Medium Not specified Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Tubulin EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 50-200

	EV160017	5/8	Homo sapiens	Cell culture supernatant	ExoQuick	Li L	2016	37%
Study summary Full title Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. All authors Li L, Li C, Wang S, Wang Z, Jiang J, Wang W, Li X, Chen J, Liu K, Li C, Zhu G. Journal Cancer Res Abstract Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient o (show more...)Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. (hide) EV-METRIC 37% (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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Hypoxic condition (1% O2) Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography ExoQuick Protein markers EV: CD81/ CD63 non-EV: Tubulin Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Hypoxic condition (1% O2) EV-producing cells SCC-9 Oral squamous cell carcinoma EV-harvesting Medium Not specified Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Tubulin EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 50-200

	EV160017	7/8	Homo sapiens	Serum	ExoQuick	Li L	2016	37%
Study summary Full title Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. All authors Li L, Li C, Wang S, Wang Z, Jiang J, Wang W, Li X, Chen J, Liu K, Li C, Zhu G. Journal Cancer Res Abstract Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient o (show more...)Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. (hide) EV-METRIC 37% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography ExoQuick Protein markers EV: CD81/ CD63 non-EV: Tubulin Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Control condition Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Tubulin EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 50-200

	EV160017	8/8	Homo sapiens	Serum	ExoQuick	Li L	2016	37%
Study summary Full title Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. All authors Li L, Li C, Wang S, Wang Z, Jiang J, Wang W, Li X, Chen J, Liu K, Li C, Zhu G. Journal Cancer Res Abstract Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient o (show more...)Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. (hide) EV-METRIC 37% (87th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin Oral squamous cell carcinoma patients Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography ExoQuick Protein markers EV: CD81/ CD63 non-EV: Tubulin Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Serum Sample Condition Oral squamous cell carcinoma patients Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Detected EV-associated proteins CD63/ CD81 Not detected contaminants Tubulin EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 50-200

	EV160002	2/3	Homo sapiens	Extruded cells (NIH3T3)	DC Sequential extrusion	Lunavat TR	2016	37%
Study summary Full title RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer. All authors Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J Journal Biomaterials Abstract To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide) EV-METRIC 37% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Extruded cells (NIH3T3) Sample origin Control condition Focus vesicles Nanovesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DC + Sequential extrusion Protein markers EV: PDGFR/ Flotillin-1/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function, New methodological development Sample Species Homo sapiens Sample Type Extruded cells (NIH3T3) Sample Condition Control condition Separation Method Density cushion Density medium Iodixanol Other Name other separation method Sequential extrusion Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Lysis buffer provided? Yes Detected EV-associated proteins PDGFR, Flotillin-1, CD9 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-200 EM EM-type Transmission-EM Image type Wide-field

	EV160002	3/3	Homo sapiens	Extruded cells (NIH3T3)	DC Sequential extrusion	Lunavat TR	2016	37%
Study summary Full title RNAi delivery by exosome-mimetic nanovesicles - Implications for targeting c-Myc in cancer. All authors Lunavat TR, Jang SC, Nilsson L, Park HT, Repiska G, Lässer C, Nilsson JA, Gho YS, Lötvall J Journal Biomaterials Abstract To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA m (show more...)To develop RNA-based therapeutics, it is crucial to create delivery vectors that transport the RNA molecule into the cell cytoplasm. Naturally released exosomes vesicles (also called "Extracellular Vesicles") have been proposed as possible RNAi carriers, but their yield is relatively small in any cell culture system. We have previously generated exosome-mimetic nanovesicles (NV) by serial extrusions of cells through nano-sized filters, which results in 100-times higher yield of extracellular vesicles. We here test 1) whether NV can be loaded with siRNA exogenously and endogenously, 2) whether the siRNA-loaded NV are taken up by recipient cells, and 3) whether the siRNA can induce functional knock-down responses in recipient cells. A siRNA against GFP was first loaded into NV by electroporation, or a c-Myc shRNA was expressed inside of the cells. The NV were efficiently loaded with siRNA with both techniques, were taken up by recipient cells, which resulted in attenuation of target gene expression. In conclusion, our study suggests that exosome-mimetic nanovesicles can be a platform for RNAi delivery to cell cytoplasm. (hide) EV-METRIC 37% (50th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Extruded cells (NIH3T3) Sample origin cMyc shRNA Focus vesicles Nanovesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DC + Sequential extrusion Protein markers EV: PDGFR/ Flotillin-1/ CD9 non-EV: Calnexin Proteomics no Show all info Study aim Function, New methodological development Sample Species Homo sapiens Sample Type Extruded cells (NIH3T3) Sample Condition cMyc shRNA Separation Method Density cushion Density medium Iodixanol Other Name other separation method Sequential extrusion Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Lysis buffer provided? Yes Detected EV-associated proteins PDGFR, Flotillin-1, CD9 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 180-200 EM EM-type Transmission-EM Image type Wide-field

	EV160012	1/8	Homo sapiens	Cell culture supernatant	dUC Filtration	Hannafon BN	2016	33%
Study summary Full title Plasma exosome microRNAs are indicative of breast cancer. All authors Hannafon BN, Trigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, Zhao ZJ, Blick KE, Dooley WC, Ding WQ. Journal Breast Cancer Res Treat Abstract BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific (show more...)BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Preparation of EDS 2h at 100,000g Separation 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) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD63 Characterization: Particle analysis NTA Report type Mean Reported size (nm) 103.3 EV concentration Yes EM EM-type Immuno-EM Proteïns CD63 Image type Close-up, Wide-field

	EV160012	2/8	Homo sapiens	Cell culture supernatant	dUC Filtration	Hannafon BN	2016	33%
Study summary Full title Plasma exosome microRNAs are indicative of breast cancer. All authors Hannafon BN, Trigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, Zhao ZJ, Blick KE, Dooley WC, Ding WQ. Journal Breast Cancer Res Treat Abstract BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific (show more...)BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS 2h at 100,000g Separation 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) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD63 Characterization: Particle analysis NTA Report type Mean Reported size (nm) 99.6 EV concentration Yes EM EM-type Immuno-EM Proteïns CD63 Image type Close-up, Wide-field

	EV160011	1/4	Homo sapiens	Cell culture supernatant	dUC ExoQuick Filtration	Hoshina S	2016	33%
Study summary Full title Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines. All authors Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H. Journal PLoS One Abstract Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Kaposi sarcoma-associated herpesvirus-infected Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + ExoQuick + Filtration Protein markers EV: Lyn/ CD63/ HSP70 non-EV: KSHV ORF45 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Kaposi sarcoma-associated herpesvirus-infected EV-producing cells BCBL1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation 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 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Lyn/ HSP70 Detected contaminants KSHV ORF45 EM EM-type Transmission-EM Image type Wide-field

	EV160011	2/4	Homo sapiens	Cell culture supernatant	dUC ExoQuick Filtration	Hoshina S	2016	33%
Study summary Full title Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines. All authors Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H. Journal PLoS One Abstract Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Kaposi sarcoma-associated herpesvirus-infected Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + ExoQuick + Filtration Protein markers EV: HSP70/ Lyn/ CD63 non-EV: KSHV ORF45 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Kaposi sarcoma-associated herpesvirus-infected EV-producing cells TY1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation 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 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ Lyn/ HSP70 Not detected contaminants KSHV ORF45 EM EM-type Transmission-EM Image type Wide-field

	EV160011	3/4	Homo sapiens	Cell culture supernatant	dUC ExoQuick Filtration	Hoshina S	2016	33%
Study summary Full title Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines. All authors Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H. Journal PLoS One Abstract Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Epstein-Barr virus-infected Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + ExoQuick + Filtration Protein markers EV: HSP70/ Lyn/ CD63 non-EV: KSHV ORF45 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Epstein-Barr virus-infected EV-producing cells LCL EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation 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 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins CD63/ HSP70/ Lyn Not detected contaminants KSHV ORF45 EM EM-type Transmission-EM Image type Wide-field

	EV160011	4/4	Homo sapiens	Cell culture supernatant	dUC ExoQuick Filtration	Hoshina S	2016	33%
Study summary Full title Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines. All authors Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H. Journal PLoS One Abstract Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + ExoQuick + Filtration Protein markers EV: HSP70/ Lyn/ CD63 non-EV: KSHV ORF45 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells Bjab EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) at >=100,000g Separation 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 Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Characterization: Protein analysis Western Blot Detected EV-associated proteins Lyn/ CD63/ HSP70 EM EM-type Transmission-EM Image type Wide-field

	EV160008	3/3	Rattus norvegicus	Cell culture supernatant	dUC	Cianciaruso C	2016	33%
Study summary Full title Primary Human and Rat β-Cells Release the Intracellular Autoantigens GAD65, IA-2, and Proinsulin in Exosomes Together With Cytokine-Induced Enhancers of Immunity. All authors Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S Journal Diabetes Abstract The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: Alix non-EV: None Proteomics yes Show all info Study aim Function, Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells INS1 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability 87.5 Separation 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 Pelleting: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix Proteomics database No

	EV160020	1/4	Homo sapiens	Cell culture supernatant	dUC	Khan MB	2016	22%
Study summary Full title Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ(1-42) secretion in SH-SY5Y neural cells. All authors Khan MB, Lang MJ, Huang MB, Raymond A, Bond VC, Shiramizu B, Powell MD. Journal J Neurovirol. Abstract In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infecti (show more...)In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aβ) and Aβ peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND. (hide) EV-METRIC 22% (49th 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin mock-transfected (empty vector expressing GFP ) Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition mock-transfected (empty vector expressing GFP ) EV-producing cells HEK293 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation 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 Pelleting: rotor type Not specified Pelleting: speed (g) 105700 Characterization: Protein analysis Western Blot Detected EV-associated proteins HSP70 Not detected EV-associated proteins CD63/ CD9/ CD81/ Nef Characterization: Particle analysis NTA Report type Mean Reported size (nm) 112 EV concentration Yes

	EV160020	2/4	Homo sapiens	Cell culture supernatant	dUC	Khan MB	2016	22%
Study summary Full title Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ(1-42) secretion in SH-SY5Y neural cells. All authors Khan MB, Lang MJ, Huang MB, Raymond A, Bond VC, Shiramizu B, Powell MD. Journal J Neurovirol. Abstract In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infecti (show more...)In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aβ) and Aβ peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND. (hide) EV-METRIC 22% (49th 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin nef expression vector transfected (pQBI-nefGFP) Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition nef expression vector transfected (pQBI-nefGFP) EV-producing cells HEK293 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation 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 Pelleting: rotor type Not specified Pelleting: speed (g) 105700 Characterization: Protein analysis Western Blot Detected EV-associated proteins HSP70/ CD81/ CD9/ CD63/ Nef Characterization: Particle analysis NTA Report type Mean Reported size (nm) 60 EV concentration Yes

	EV160006	1/1	Homo sapiens	Cell culture supernatant	dUC Filtration	Holder B	2016	22%
Study summary Full title Macrophage Exosomes Induce Placental Inflammatory Cytokines: A Novel Mode of Maternal-Placental Messaging. All authors Holder B, Jones T, Sancho Shimizu V, Rice TF, Donaldson B, Bouqueau M, Forbes K, Kampmann B Journal Traffic Abstract During pregnancy, the placenta forms the interface between mother and fetus. Highly controlled regul (show more...)During pregnancy, the placenta forms the interface between mother and fetus. Highly controlled regulation of trans-placental trafficking is therefore essential for the healthy development of the growing fetus. Extracellular vesicle-mediated transfer of protein and nucleic acids from the human placenta into the maternal circulation is well documented; the possibility that this trafficking is bi-directional has not yet been explored but could affect placental function and impact on the fetus.We hypothesized that the ability of the placenta to respond to maternal inflammatory signals is mediated by the interaction of maternal immune cell exosomes with placental trophoblast. Utilizing the BeWo cell line and whole placental explants, we demonstrated that the human placenta internalizes macrophage-derived exosomes in a time- and dose-dependent manner. This uptake was via clathrin-dependent endocytosis. Furthermore, macrophage exosomes induced release of proinflammatory cytokines by the placenta. Taken together, our data demonstrates that exosomes are actively transported into the human placenta and that exosomes from activated immune cells modulate placental cytokine production. This represents a novel mechanism by which immune cells can signal to the placental unit, potentially facilitating responses to maternal inflammation and infection, and thereby preventing harm to the fetus. (hide) EV-METRIC 22% (49th 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin PMA-stimulated Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Protein markers EV: Alix/ CD81 non-EV: Calnexin Proteomics no Show all info Study aim Function, Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition PMA-stimulated EV-producing cells THP-1 EV-harvesting Medium EV-depleted serum Preparation of EDS Commercial EDS Separation 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 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Lysis buffer provided? Yes Detected EV-associated proteins Alix, CD81 Not detected contaminants Calnexin Characterization: Particle analysis NTA Report type Mean Reported size (nm) 137.6+-18.5 EV concentration Yes Extra information I think that recording how EVs were labelled (including controls), and how EVs were stored prior to functional analysis (e.g. fresh/frozen) would be a good addition to this

	EV160017	3/8	Homo sapiens	Cell culture supernatant	ExoQuick	Li L	2016	17%
Study summary Full title Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. All authors Li L, Li C, Wang S, Wang Z, Jiang J, Wang W, Li X, Chen J, Liu K, Li C, Zhu G. Journal Cancer Res Abstract Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient o (show more...)Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. (hide) EV-METRIC 17% (43rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin HIF-1/HIF-2 alpha knockdown + Hypoxia Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography ExoQuick Protein markers EV: non-EV: Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition HIF-1/HIF-2 alpha knockdown + Hypoxia EV-producing cells Cal-27 Oral squamous cell carcinoma EV-harvesting Medium Not specified Separation Method Commercial kit ExoQuick Protein Concentration Method Not Determined EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 50-200

	EV160017	6/8	Homo sapiens	Cell culture supernatant	ExoQuick	Li L	2016	17%
Study summary Full title Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype. All authors Li L, Li C, Wang S, Wang Z, Jiang J, Wang W, Li X, Chen J, Liu K, Li C, Zhu G. Journal Cancer Res Abstract Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient o (show more...)Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. (hide) EV-METRIC 17% (43rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin HIF-1/HIF-2 alpha knockdown + Hypoxia Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography ExoQuick Protein markers EV: non-EV: Proteomics no Show all info Study aim Function/Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition HIF-1/HIF-2 alpha knockdown + Hypoxia EV-producing cells SCC-9 Oral squamous cell carcinoma EV-harvesting Medium Not specified Separation Method Commercial kit ExoQuick Protein Concentration Method Not Determined EM EM-type Scanning-EM Image type Close-up, Wide-field Report size (nm) 50-200

	EV160013	2/3	Homo sapiens	Serum	dUC	Dong L	2016	14%
Study summary Full title Circulating Long RNAs in Serum Extracellular Vesicles: Their Characterization and Potential Application as Biomarkers for Diagnosis of Colorectal Cancer. All authors Dong L, Lin W, Qi P, Xu MD, Wu X, Ni S, Huang D, Weng WW, Tan C, Sheng W, Zhou X, Du X. Journal Cancer Epidemiol Biomarkers Prev Abstract BACKGROUND: Long noncoding RNA (lncRNA) and mRNAs are long RNAs (≥200 nucleotides) compared with m (show more...)BACKGROUND: Long noncoding RNA (lncRNA) and mRNAs are long RNAs (≥200 nucleotides) compared with miRNAs. In blood, long RNAs may be protected by serum extracellular vesicles, such as apoptotic bodies (AB), microvesicles (MV), and exosomes (EXO). They are potential biomarkers for identifying cancer. METHODS: Sera from 76 preoperative colorectal cancer patients, 76 age- and sex-matched healthy subjects, and 20 colorectal adenoma patients without colorectal cancer were collected. We investigated the distribution of long RNAs into the three vesicles. Seventy-nine cancer-related long RNAs were chosen and detected using qPCR. RESULTS: The quantity of long RNA has varying distribution among three subtypes of extracellular vesicles in serum. Most mRNA and lncRNA genes had higher quantity in EXOs than that in ABs and MVs, whereas MVs contain lowest quantity. We investigated 79 long RNAs chosen from The Cancer Genome Atlas and the LncRNADisease database in the sera of healthy patients, and those with colorectal cancer. In the training and test sets, the AUCs were 0.936 and 0.877, respectively. The AUC of total serum RNA was lower (0.857) than that of exosomal RNA in the same samples (0.936). CONCLUSION: The present study shows that exosomal mRNAs and lncRNAs in serum could be used as biomarkers to detect colorectal cancer. (hide) EV-METRIC 14% (63rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin colorectal cancer Focus vesicles (shedding) microvesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Sample Condition colorectal cancer Separation 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 Pelleting: rotor type Not specified Pelleting: speed (g) 12000 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 75-465 EV concentration Yes EM EM-type Transmission-EM Image type Close-up

	EV160013	3/3	Homo sapiens	Serum	dUC	Dong L	2016	14%
Study summary Full title Circulating Long RNAs in Serum Extracellular Vesicles: Their Characterization and Potential Application as Biomarkers for Diagnosis of Colorectal Cancer. All authors Dong L, Lin W, Qi P, Xu MD, Wu X, Ni S, Huang D, Weng WW, Tan C, Sheng W, Zhou X, Du X. Journal Cancer Epidemiol Biomarkers Prev Abstract BACKGROUND: Long noncoding RNA (lncRNA) and mRNAs are long RNAs (≥200 nucleotides) compared with m (show more...)BACKGROUND: Long noncoding RNA (lncRNA) and mRNAs are long RNAs (≥200 nucleotides) compared with miRNAs. In blood, long RNAs may be protected by serum extracellular vesicles, such as apoptotic bodies (AB), microvesicles (MV), and exosomes (EXO). They are potential biomarkers for identifying cancer. METHODS: Sera from 76 preoperative colorectal cancer patients, 76 age- and sex-matched healthy subjects, and 20 colorectal adenoma patients without colorectal cancer were collected. We investigated the distribution of long RNAs into the three vesicles. Seventy-nine cancer-related long RNAs were chosen and detected using qPCR. RESULTS: The quantity of long RNA has varying distribution among three subtypes of extracellular vesicles in serum. Most mRNA and lncRNA genes had higher quantity in EXOs than that in ABs and MVs, whereas MVs contain lowest quantity. We investigated 79 long RNAs chosen from The Cancer Genome Atlas and the LncRNADisease database in the sera of healthy patients, and those with colorectal cancer. In the training and test sets, the AUCs were 0.936 and 0.877, respectively. The AUC of total serum RNA was lower (0.857) than that of exosomal RNA in the same samples (0.936). CONCLUSION: The present study shows that exosomal mRNAs and lncRNAs in serum could be used as biomarkers to detect colorectal cancer. (hide) EV-METRIC 14% (63rd 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin colorectal cancer Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Serum Sample Condition colorectal cancer Separation 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 Not specified Pelleting: speed (g) 120000 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 45-205 EM EM-type Transmission-EM Image type Close-up

	EV160003	1/1	Mus musculus	Cell culture supernatant	dUC	Morales-Kastresana A	2016	14%
Study summary Full title Labeling Extracellular Vesicles for Nanoscale Flow Cytometry. All authors Morales-Kastresana A, Telford B, Musich TA, McKinnon K, Clayborne C, Braig Z, Rosner A, Demberg T, Watson DC, Karpova TS, Freeman GJ, DeKruyff RH, Pavlakis GN, Terabe M, Robert-Guroff M, Berzofsky JA, Jones JC Journal Sci Rep Abstract Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that ar (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles, are 30-800 nm vesicles that are released by most cell types, as biological packages for intercellular communication. Their importance in cancer and inflammation makes EVs and their cargo promising biomarkers of disease and cell-free therapeutic agents. Emerging high-resolution cytometric methods have created a pressing need for efficient fluorescent labeling procedures to visualize and detect EVs. Suitable labels must be bright enough for one EV to be detected without the generation of label-associated artifacts. To identify a strategy that robustly labels individual EVs, we used nanoFACS, a high-resolution flow cytometric method that utilizes light scattering and fluorescence parameters along with sample enumeration, to evaluate various labels. Specifically, we compared lipid-, protein-, and RNA-based staining methods and developed a robust EV staining strategy, with the amine-reactive fluorescent label, 5-(and-6)-Carboxyfluorescein Diacetate Succinimidyl Ester, and size exclusion chromatography to remove unconjugated label. By combining nanoFACS measurements of light scattering and fluorescence, we evaluated the sensitivity and specificity of EV labeling assays in a manner that has not been described for other EV detection methods. Efficient characterization of EVs by nanoFACS paves the way towards further study of EVs and their roles in health and disease. (hide) EV-METRIC 14% (39th 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC Adj. k-factor 156.9 (pelleting) / 41.45 (washing) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim New methodological development, Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells DC2.4 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Separation 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 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 156.9 Wash: time (min) 70 Wash: Rotor Type TLA-120.1 Wash: speed (g) 100000 Wash: adjusted k-factor 41.45 Protein Concentration Method BCA Protein Concentration 1.186 Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 80-180 EV concentration Yes Particle yield 4.8E+11 particles/ml start sample Particle analysis: flow cytometry Flow cytometer type Beckman Astrios EQ Hardware adjustment NanoFACS system Calibration bead size 0.1;0.2;0.5

	EV160012	3/8	Homo sapiens	Cell culture supernatant	dUC Filtration	Hannafon BN	2016	11%
Study summary Full title Plasma exosome microRNAs are indicative of breast cancer. All authors Hannafon BN, Trigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, Zhao ZJ, Blick KE, Dooley WC, Ding WQ. Journal Breast Cancer Res Treat Abstract BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific (show more...)BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. (hide) EV-METRIC 11% (26th 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 Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = differential ultracentrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography dUC + Filtration Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells MCF10A EV-harvesting Medium EV-depleted medium Preparation of EDS 2h at 100,000g Separation 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) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins CD63 Characterization: Particle analysis NTA Report type Mean Reported size (nm) 126.4 EV concentration Yes

	EV160020	3/4	Homo sapiens	Blood plasma	dUC	Khan MB	2016	0%
Study summary Full title Nef exosomes isolated from the plasma of individuals with HIV-associated dementia (HAD) can induce Aβ(1-42) secretion in SH-SY5Y neural cells. All authors Khan MB, Lang MJ, Huang MB, Raymond A, Bond VC, Shiramizu B, Powell MD. Journal J Neurovirol. Abstract In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infecti (show more...)In the era of combined antiretroviral therapy (CART), many of the complications due to HIV-1 infection have diminished. One exception is HIV-associated neurocognitive disorder (HAND). HAND is a spectrum of disorders in cognitive function that ranges from asymptomatic disease to severe dementia (HAD). The milder form of HAND has actually remained the same or slightly increased in prevalence in the CART era. Even in individuals who have maintained undetectable HIV RNA loads, viral proteins such as Nef and Tat can continue to be expressed. In this report, we show that Nef protein and nef messenger RNA (mRNA) are packaged into exosomes that remain in circulation in patients with HAD. Plasma-derived Nef exosomes from patients with HAD have the ability to interact with the neuroblastoma cell line SH-SY5Y and deliver nef mRNA. The mRNA can induce expression of Nef in target cells and subsequently increase expression and secretion of beta-amyloid (Aβ) and Aβ peptides. Increase secretion of amyloid peptide could contribute to cognitive impairment seen in HAND. (hide) EV-METRIC 0% (median: 14% of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis

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