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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210117	3/3	Homo sapiens	SK-N-BE(2)-C	(d)(U)C	Helge Haug, Bjørn	2015	33%
Study summary Full title Exosome-like Extracellular Vesicles from MYCN-amplified Neuroblastoma Cells Contain Oncogenic miRNAs All authors Bjørn Helge Haug, Øyvind H Hald, Peter Utnes, Sarah A Roth, Cecilie Løkke, Trond Flægstad, Christer Einvik Journal Anticancer Res Abstract Background: In recent years, evidence has accumulated indicating that both normal and cancer cells c (show more...)Background: In recent years, evidence has accumulated indicating that both normal and cancer cells communicate via the release and delivery of macromolecules packed into extracellular membrane vesicles. Materials and methods: We isolated nano-sized extracellular vesicles from MYCN-amplified neuroblastoma cell lines using ultracentrifugation and exosome precipitation (Exoquick) protocols. These vesicles were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis and western blotting. Exosomal miRNA profiles were obtained using a reverse transcription-polymerase chain reaction (RT-PCR) ready-to-use panel measuring a total of 742 miRNAs. Results: In this study, we showed that MYCN-amplified neuroblastoma cell lines secrete populations of miRNAs inside small exosome-like vesicular particles. These particles were shown to be taken-up by recipient cells. By profiling the miRNA content, we demonstrated high expression of a group of established oncomirs in exosomes from two MYCN-amplified neuroblastoma cell lines. Despite the fact that other studies have demonstrated the ability of exosomal miRNAs both to repress mRNA targets and to stimulate Toll-like receptor-8 (TLR8) signaling in recipient cells, we did not observe these effects with exosomes from MYCN-amplified neuroblastoma cells. However, functional enrichment analysis reveals that mRNA targets of highly expressed exosomal miRNAs are associated with a range of cellular and molecular functions related to cell growth and cell death. Conclusion: MYCN-amplified neuroblastoma cell lines secrete exosome-like particles containing oncogenic miRNAs. This work showed for the first time that neuroblastoma cells secrete exosome-like particles containing miRNAs with potential roles in cancer progression. These findings indicate a new way for MYCN-amplified neuroblastoma cells to interact with the tumor environment. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: TSG101/ CD63/ CD9 non-EV: actin/ N-myc/ GRP78 Proteomics no Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SK-N-BE(2)-C EV-harvesting Medium EV-depleted medium;Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask) Preparation of EDS overnight (16h) at >=100,000g + filtration 0.2 µm Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Not specified Pelleting: speed (g) 110 000 Characterization: Protein analysis Protein Concentration Method Lowry-based assay Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101 Not detected contaminants actin/ N-myc/ GRP78 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer) Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No

	EV160016	2/3	Homo sapiens	DLD-1	(d)(U)C UF Filtration	Cha DJ	2015	33%
Study summary Full title KRAS-dependent sorting of miRNA to exosomes All authors Cha DJ, Franklin JL, Dou Y, Liu Q, Higginbotham JN, Demory Beckler M, Weaver AM, Vickers K, Prasad N, Levy S, Zhang B, Coffey RJ, Patton JG. Journal Elife Abstract Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor mi (show more...)Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the... (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C UF Filtration Protein markers EV: "Flotillin1/ TSG101/ HSP70/ KRAS/ EGFR/ RAP1/ SRC/ LYN/ ITGB1/ ITGA2/ ITGAV/ ITGB4/ EPHA2/ EPS8/ CTNNA" non-EV: VDAC Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells DLD-1 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Not specified Pelleting: speed (g) 150000 Wash: time (min) 180 Wash: Rotor Type Not specified Wash: speed (g) 150000 Filtration steps 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Not specified Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins "Flotillin1/ TSG101/ HSP70/ KRAS/ EGFR/ RAP1/ SRC/ LYN/ ITGB1/ ITGA2/ ITGAV/ ITGB4/ EPHA2/ EPS8/ CTNNA" Not detected contaminants VDAC Proteomics database No Characterization: RNA analysis RNA analysis Type "(RT)(q)PCR;RNA sequencing" Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV150104	2/4	Homo sapiens	Urine	DC (d)(U)C	Pocsfalvi G	2015	33%
Study summary Full title Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. All authors Pocsfalvi G, Raj DA, Fiume I, Vilasi A, Trepiccione F, Capasso G. Journal Proteomics Clin Appl Abstract PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathop (show more...)PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin autosomal dominant polycystic kidney disease (late stage) Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DC (d)(U)C Protein markers EV: TSG101/ AQP2/ PKD2/ PKD1/ Alix/ CD9/ NHE3 non-EV: / Uromodulin/ Albumin Proteomics yes EV density (g/ml) Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Density gradient Only used for validation of main results Yes Type Number of initial discontinuous layers Lowest density fraction Highest density fraction Total gradient volume, incl. sample (mL) Sample volume (mL) Orientation Rotor type Speed (g) Duration (min) Fraction volume (mL) Fraction processing Pelleting: volume per fraction Pelleting: duration (min) Pelleting: rotor type Pelleting: speed (g) Pelleting-wash: volume per pellet (mL) Pelleting-wash: duration (min) Pelleting-wash: speed (g) Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ NHE3/ AQP2/ CD9/ TSG101 Not detected EV-associated proteins PKD1/ PKD2 Detected contaminants Not detected contaminants Albumin/ Uromodulin Proteomics database No Characterization: Lipid analysis No EM EM-type EV concentration

	EV150104	3/4	Homo sapiens	Urine	DC (d)(U)C	Pocsfalvi G	2015	33%
Study summary Full title Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. All authors Pocsfalvi G, Raj DA, Fiume I, Vilasi A, Trepiccione F, Capasso G. Journal Proteomics Clin Appl Abstract PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathop (show more...)PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin autosomal dominant polycystic kidney disease (early stage) Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DC (d)(U)C Protein markers EV: TSG101/ AQP2/ PKD2/ PKD1/ Alix/ CD9/ NHE3 non-EV: / Uromodulin/ Albumin Proteomics yes EV density (g/ml) Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Density gradient Only used for validation of main results Yes Type Number of initial discontinuous layers Lowest density fraction Highest density fraction Total gradient volume, incl. sample (mL) Sample volume (mL) Orientation Rotor type Speed (g) Duration (min) Fraction volume (mL) Fraction processing Pelleting: volume per fraction Pelleting: duration (min) Pelleting: rotor type Pelleting: speed (g) Pelleting-wash: volume per pellet (mL) Pelleting-wash: duration (min) Pelleting-wash: speed (g) Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ NHE3/ AQP2/ CD9/ TSG101 Not detected EV-associated proteins PKD1/ PKD2 Detected contaminants Not detected contaminants Albumin/ Uromodulin Proteomics database No Characterization: Lipid analysis No EM EM-type EV concentration

	EV150104	4/4	Homo sapiens	Urine	DC (d)(U)C	Pocsfalvi G	2015	33%
Study summary Full title Urinary extracellular vesicles as reservoirs of altered proteins during the pathogenesis of polycystic kidney disease. All authors Pocsfalvi G, Raj DA, Fiume I, Vilasi A, Trepiccione F, Capasso G. Journal Proteomics Clin Appl Abstract PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathop (show more...)PURPOSE: Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EXPERIMENTAL DESIGN: EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. RESULTS: Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin autosomal dominant polycystic kidney disease (tolvaptan treatment) Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DC (d)(U)C Protein markers EV: TSG101/ AQP2/ PKD2/ PKD1/ Alix/ CD9/ NHE3 non-EV: / Uromodulin/ Albumin Proteomics yes EV density (g/ml) Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type Not specified Pelleting: speed (g) 200000 Density gradient Only used for validation of main results Yes Type Number of initial discontinuous layers Lowest density fraction Highest density fraction Total gradient volume, incl. sample (mL) Sample volume (mL) Orientation Rotor type Speed (g) Duration (min) Fraction volume (mL) Fraction processing Pelleting: volume per fraction Pelleting: duration (min) Pelleting: rotor type Pelleting: speed (g) Pelleting-wash: volume per pellet (mL) Pelleting-wash: duration (min) Pelleting-wash: speed (g) Density cushion Density medium Sucrose Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ NHE3/ AQP2/ CD9/ TSG101 Not detected EV-associated proteins PKD1/ PKD2 Detected contaminants Not detected contaminants Albumin/ Uromodulin Proteomics database No Characterization: Lipid analysis No EM EM-type EV concentration

	EV150013	1/1	Mus musculus	DNF	(d)(U)C ExoQuick Filtration	Zhu Y	2015	33%
Study summary Full title A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor-Associated Macrophages All authors Zhu Y, Chen X, Pan Q, Wang Y, Su S, Jiang C, Li Y, Xu N, Wu L, Lou X, Liu S Journal J Proteome Res Abstract Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extrac (show more...)Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin DNF Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick Filtration Protein markers EV: Alix/ TSG101/ HSP90 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Omics Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells DNF EV-harvesting Medium serum free Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Density gradient Density medium DNF Pelleting: adjusted k-factor DNF Pelleting-wash: rotor type DNF Pelleting-wash: adjusted k-factor DNF Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ HSP90/ TSG101 Detected contaminants Cell organelle protein Proteomics database DNF Characterization: Lipid analysis DNF Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field EV concentration DNF

	EV150033	1/1	Mus musculus	NAY	(d)(U)C	DeClercq V	2015	33%
Study summary Full title Fatty acids increase adiponectin secretion through both classical and exosome pathways All authors DeClercq V, D'Eon B, McLeod RS Journal Biochim Biophys Act Cell Biol L Abstract Little is known about the effects of fatty acids on adiponectin oligomer assembly and trafficking. T (show more...)Little is known about the effects of fatty acids on adiponectin oligomer assembly and trafficking. The aim of this study was to examine the effects of different fatty acids on adiponectin transport and secretion in differentiated 3T3-L1 adipocytes. Subcellular fractionation and immunofluorescence microscopy revealed that the majority of cellular adiponectin was located in the endoplasmic reticulum (ER). Adiponectin secretion was increased by treatment with fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and several fatty acids changed the cellular localization of adiponectin. Adiponectin secretion has been shown to be altered by ER stress and interactions with ER chaperone proteins. However these mechanisms were not influenced by fatty acids, suggesting that alternative mechanisms must be responsible for the increased secretion of adiponectin observed with fatty acid treatment. Secretion of adiponectin was blocked by Brefeldin A, but we identified a minor pool of adiponectin that could be secreted from beyond the Brefeldin A block. Exosomes appeared to contribute to a minor amount of adiponectin secreted from the cell, and exosome release was increased by treatment with DHA. These data suggest that the ER is an important site of adiponectin accumulation and that treatment with long chain omega-3 fatty acids increases adiponectin release. Furthermore, the secretory pathway of adiponectin is complex, involving both the classical ER-Golgi pathway as well as unconventional secretory mechanisms such as an exosome-mediated pathway. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ TSG101 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biogenesis/Sorting Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis None

	EV150032	1/1	Bos bovis	Milk	(d)(U)C Filtration	Wolf T	2015	33%
Study summary Full title The Intestinal Transport of Bovine Milk Exosomes Is Mediated by Endocytosis in Human Colon Carcinoma Caco-2 Cells and Rat Small Intestinal IEC-6 Cells All authors Wolf T, Baier SR, Zempleni J Journal J Nutr Abstract BACKGROUND: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs i (show more...)BACKGROUND: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs in tissues and body fluids is encapsulated in exosomes, thereby conferring protection against degradation and a pathway for intestinal transport. MicroRNAs in cow milk are bioavailable in humans. OBJECTIVE: This research assessed the transport mechanism of bovine milk exosomes, and therefore microRNAs, in human and rodent intestinal cells. METHODS: The intestinal transport of bovine milk exosomes and microRNAs was assessed using fluorophore-labeled bovine milk exosomes in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. Transport kinetics and mechanisms were characterized using dose-response studies, inhibitors of vesicle transport, carbohydrate competitors, proteolysis of surface proteins on cells and exosomes, and transepithelial transport in transwell plates. RESULTS: Exosome transport exhibited saturation kinetics at 37°C [Michaelis constant (Km) = 55.5 ± 48.6 ?g exosomal protein/200 ?L of media; maximal transport rate = 0.083 ± 0.057 ng of exosomal protein · 81,750 cells(-1) · h(-1)] and decreased by 64% when transport was measured at 4°C, consistent with carrier-mediated transport in Caco-2 cells. Exosome uptake decreased by 61-85% under the following conditions compared with controls in Caco-2 cells: removal of exosome and cell surface proteins by proteinase K, inhibition of endocytosis and vesicle trafficking by synthetic inhibitors, and inhibition of glycoprotein binding by carbohydrate competitors. When milk exosomes, at a concentration of 5 times the Km, were added to the upper chamber in transwell plates, Caco-2 cells accumulated miR-29b and miR-200c in the lower chamber, and reverse transport was minor. Transport characteristics were similar in IEC-6 cells and Caco-2 cells, except that substrate affinity and transporter capacity were lower and higher, respectively. CONCLUSION: The uptake of bovine milk exosomes is mediated by endocytosis and depends on cell and exosome surface glycoproteins in human and rat intestinal cells. (hide) EV-METRIC 33% (53rd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 176.2 (pelleting) Protein markers EV: CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Function Sample Species Bos bovis Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type F37L-8x100 Pelleting: adjusted k-factor 176.2 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV150054	1/1	Mus musculus	NAY	(d)(U)C Filtration	Wang X	2015	33%
Study summary Full title Exosomal miR-223 Contributes to Mesenchymal Stem Cell-Elicited Cardioprotection in Polymicrobial Sepsis All authors Wang X, Gu H, Qin D, Yang L, Huang W, Essandoh K, Wang Y, Caldwell CC, Peng T, Zingarelli B, Fan GC Journal Sci Rep Abstract Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However (show more...)Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 206.6 (pelleting) Protein markers EV: CD81/ CD63 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 45Ti Pelleting: adjusted k-factor 206.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ CD81 Characterization: Particle analysis DLS

	EV150030	1/1	Homo sapiens	Urine	(d)(U)C	Perez-Hernandez J	2015	33%
Study summary Full title Increased Urinary Exosomal MicroRNAs in Patients with Systemic Lupus Erythematosus All authors Perez-Hernandez J, Forner MJ, Pinto C, Chaves FJ, Cortes R, Redon J Journal PLoS One Abstract There is increased interest in using microRNAs (miRNAs) as biomarkers in different diseases. Present (show more...)There is increased interest in using microRNAs (miRNAs) as biomarkers in different diseases. Present in body fluids, it is controversial whether or not they are mainly enclosed in exosomes, thus we studied if urinary miRNAs are concentrated inside exosomes and if the presence of systemic lupus erythematosus with or without lupus nephritis modifies their distribution pattern. We quantified specific miRNAs in urine of patients with systemic lupus erythematosus (n = 38) and healthy controls (n = 12) by quantitative reverse-transcription PCR in cell-free urine, exosome-depleted supernatant and exosome pellet obtained by ultracentrifugation. In control group, miR-335 (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 27.21 (pelleting) Protein markers EV: TSG101/ CD9 non-EV: Cell organelle protein Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 70.1Ti Pelleting: adjusted k-factor 27.21 Wash: volume per pellet (ml) 2 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ TSG101 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up, Wide-field

	EV150029	1/1	Homo sapiens	Urine	(d)(U)C Filtration UF	Øverbye A	2015	33%
Study summary Full title Identification of prostate cancer biomarkers in urinary exosomes All authors Øverbye A, Skotland T, Koehler CJ, Thiede B, Seierstad T, Berge V, Sandvig K, Llorente A Journal Oncotarget Abstract Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-spe (show more...)Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-specific molecules can be found in exosomes isolated from biological fluids. We have here investigated the proteome of urinary exosomes by using mass spectrometry to identify proteins differentially expressed in prostate cancer patients compared to healthy male controls. In total, 15 control and 16 prostate cancer samples of urinary exosomes were analyzed. Importantly, 246 proteins were differentially expressed in the two groups. The majority of these proteins (221) were up-regulated in exosomes from prostate cancer patients. These proteins were analyzed according to specific criteria to create a focus list that contained 37 proteins. At 100% specificity, 17 of these proteins displayed individual sensitivities above 60%. Even though several of these proteins showed high sensitivity and specificity for prostate cancer as individual biomarkers, combining them in a multi-panel test has the potential for full differentiation of prostate cancer from non-disease controls. The highest sensitivity, 94%, was observed for transmembrane protein 256 (TM256; chromosome 17 open reading frame 61). LAMTOR proteins were also distinctly enriched with very high specificity for patient samples. TM256 and LAMTOR1 could be used to augment the sensitivity to 100%. Other prominent proteins were V-type proton ATPase 16 kDa proteolipid subunit (VATL), adipogenesis regulatory factor (ADIRF), and several Rab-class members and proteasomal proteins. In conclusion, this study clearly shows the potential of using urinary exosomes in the diagnosis and clinical management of prostate cancer. (hide) EV-METRIC 33% (65th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Urine Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration UF Adj. k-factor 156.9 (pelleting) / 276.6 (washing) Protein markers EV: CD81/ TSG101/ CD9 non-EV: Tamm-Horsfall glycoprotein Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Urine Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: Rotor Type SW40 Wash: adjusted k-factor 276.6 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD81/ CD9/ TSG101 Detected contaminants Tamm-Horsfall glycoprotein Characterization: Particle analysis EM EM-type immune EM EM protein CD63 Image type Wide-field

	EV150007	6/10	Homo sapiens	NAY	(d)(U)C DG Filtration	Lobb RJ	2015	33%
Study summary Full title Optimized exosome isolation protocol for cell culture supernatant and human plasma All authors Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A Journal J Extracell Vesicles Abstract Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 157.1 (pelleting) / 157.1 (washing) Protein markers EV: TSG101/ Flotilin1 non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Wash: volume per pellet (ml) 1 Wash: Rotor Type 50.2Ti Wash: adjusted k-factor 157.1 Density gradient Lowest density fraction 5 Highest density fraction 40 Orientation Top-down Rotor type 50.2Ti Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Flotilin1/ TSG101 Characterization: Particle analysis TRPS

	EV150028	1/1	Homo sapiens	NAY	(d)(U)C Filtration	Koch R	2015	33%
Study summary Full title Nuclear trapping through inhibition of exosomal export by indomethacin increases cytostatic efficacy of doxorubicin and pixantrone All authors Koch R, Aung T, Vogel D, Chapuy B, Wenzel D, Becker S, Sinzig U, Venkataramani V, von Mach T, Jacob R, Truemper L, Wulf GG Journal Clin Cancer Res Abstract PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with agg (show more...)PURPOSE: Although R-CHOP-based immunochemotherapy cures significant proportions of patients with aggressive B-cell lymphoma, tumor cell susceptibility to chemotherapy varies, with mostly fatal outcome in cases of resistant disease. We and others have shown before that export of cytostatic drugs contributes to drug resistance. Now we provide a novel approach to overcome exosome-mediated drug resistance in aggressive B-cell lymphomas. EXPERIMENTAL DESIGN: We used well-established centrifugation protocols to purify exosomes from DLBCL cell lines and detected anthracyclines using FACS and HPLC. We used shRNA knockdown of ABCA3 to determine ABCA3 dependence of chemotherapy susceptibility and monitored ABCA3 expression after indomethacin treatment using qPCR. Finally, we established an in vivo assay using a chorioallantoic membrane (CAM) assay to determine the synergy of anthracycline and indomethacin treatment. RESULTS: We show increased efficacy of the anthracycline doxorubicin and the anthracenedione pixantrone by suppression of exosomal drug resistance with indomethacin. B-cell lymphoma cells in vitro efficiently extruded doxorubicin and pixantrone, in part compacted in exosomes. Exosomal biogenesis was critically dependent on the expression of the ATP-transporter A3 (ABCA3). Genetic or chemical depletion of ABCA3 augmented intracellular retention of both drugs and shifted the subcellular drug accumulation to prolonged nuclear retention. Indomethacin increased the cytostatic efficacy of both drugs against DLBCL cell lines in vitro and in vivo in a CAM assay. CONCLUSIONS: We propose pretreatment with indomethacin toward enhanced antitumor efficacy of anthracyclines and anthracenediones. Clin Cancer Res; 1-10. ©2015 AACR. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 130.7 (pelleting) Protein markers EV: CD63/ CD81/ GAPDH/ ADAM10/ Flotillin2/ CD9 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 130.7 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9/ "ADAM10/ Flotillin2/ GAPDH" ELISA Antibody details provided? No Detected EV-associated proteins "ADAM10/ Flotillin2/ GAPDH" Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV150041	1/1	Homo sapiens	NAY	(d)(U)C	Lamichhane TN	2015	33%
Study summary Full title Exogenous DNA Loading into Extracellular Vesicles via Electroporation is Size-Dependent and Enables Limited Gene Delivery All authors Lamichhane TN, Raiker RS, Jay SM Journal Mol Pharm Abstract Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delive (show more...)Extracellular vesicles (EVs) hold immense promise for utilization as biotherapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Specifically, EVs have been identified as natural carriers of nucleic acids, sparking interest in their use for gene therapy and RNA interference applications. So far, small RNAs (siRNA and miRNA) have been successfully loaded into EVs for a variety of delivery applications, but the potential use of EVs for DNA delivery has scarcely been explored. Here, we report that exogenous linear DNA can be associated with EVs via electroporation in quantities sufficient to yield an average of hundreds of DNA molecules per vesicle. We determined that loading efficiency and capacity of DNA in EVs is dependent on DNA size, with linear DNA molecules less than 1000 bp in length being more efficiently associated with EVs compared to larger linear DNAs and plasmid DNAs using this approach. We further showed that EV size is also determinant with regard to DNA loading, as larger microvesicles encapsulated more linear and plasmid DNA than smaller, exosome-like EVs. Additionally, we confirmed the ability of EVs to transfer foreign DNA loaded via electroporation into recipient cells, although functional gene delivery was not observed. These results establish critical parameters that inform the potential use of EVs for gene therapy and, in agreement with other recent results, suggest that substantial barriers must be overcome to establish EVs as broadly applicable DNA delivery vehicles. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 156.9 (pelleting) / 156.9 (washing) Protein markers EV: Alix non-EV: GAPDH Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Wash: volume per pellet (ml) 29 Wash: Rotor Type 70Ti Wash: adjusted k-factor 156.9 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix Detected contaminants GAPDH Characterization: Particle analysis NTA

	EV150014	1/2	Homo sapiens	NAY	(d)(U)C Filtration SEC UF	Grasso L	2015	33%
Study summary Full title Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy All authors Grasso L, Wyss R, Weidenauer L, Thampi A, Demurtas D, Prudent M, Lion N, Vogel H Journal Anal Bioanal Chem Abstract We report on a generic method to detect and identify the molecular profile of exosomes either derive (show more...)We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration SEC UF Protein markers EV: CD44/ EpCAM/ CD63/ CD9/ CD24 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD44/ CD24/ EpCAM ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD44/ CD24/ EpCAM Characterization: Particle analysis DLS EM EM-type cryo EM Image type Close-up

	EV150014	2/2	Homo sapiens	Blood plasma	(d)(U)C Filtration	Grasso L	2015	33%
Study summary Full title Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy All authors Grasso L, Wyss R, Weidenauer L, Thampi A, Demurtas D, Prudent M, Lion N, Vogel H Journal Anal Bioanal Chem Abstract We report on a generic method to detect and identify the molecular profile of exosomes either derive (show more...)We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: CD44/ CD63/ CD9/ CD24 non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD44/ CD24 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD9/ CD44/ CD24 Characterization: Particle analysis DLS EM EM-type cryo EM Image type Close-up

	EV150023	1/1	Homo sapiens	Blood plasma	(d)(U)C Filtration	Frühbeis C	2015	33%
Study summary Full title Physical exercise induces rapid release of small extracellular vesicles into the circulation All authors Frühbeis C, Helmig S, Tug S, Simon P, Krämer-Albers EM Journal J Extracell Vesicles Abstract Cells secrete extracellular vesicles (EVs) by default and in response to diverse stimuli for the pur (show more...)Cells secrete extracellular vesicles (EVs) by default and in response to diverse stimuli for the purpose of cell communication and tissue homeostasis. EVs are present in all body fluids including peripheral blood, and their appearance correlates with specific physiological and pathological conditions. Here, we show that physical activity is associated with the release of nano-sized EVs into the circulation. Healthy individuals were subjected to an incremental exercise protocol of cycling or running until exhaustion, and EVs were isolated from blood plasma samples taken before, immediately after and 90 min after exercise. Small EVs with the size of 100-130 nm, that carried proteins characteristic of exosomes, were significantly increased immediately after cycling exercise and declined again within 90 min at rest. In response to treadmill running, elevation of small EVs was moderate but appeared more sustained. To delineate EV release kinetics, plasma samples were additionally taken at the end of each increment of the cycling exercise protocol. Release of small EVs into the circulation was initiated in an early phase of exercise, before the individual anaerobic threshold, which is marked by the rise of lactate. Taken together, our study revealed that exercise triggers a rapid release of EVs with the characteristic size of exosomes into the circulation, initiated in the aerobic phase of exercise. We hypothesize that EVs released during physical activity may participate in cell communication during exercise-mediated adaptation processes that involve signalling across tissues and organs. (hide) EV-METRIC 33% (66th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 90.14 (pelleting) Protein markers EV: TSG101/ HSP70/ Flotilin1 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA55 Pelleting: adjusted k-factor 90.14 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins Flotilin1/ HSP70/ TSG101 Characterization: Particle analysis NTA

	EV150022	1/1	Homo sapiens	NAY	(d)(U)C	Endo-Munoz L	2015	33%
Study summary Full title Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis All authors Endo-Munoz L, Cai N, Cumming A, Macklin R, Merida de Long L, Topkas E, Mukhopadhyay P, Hill M, Saunders NA Journal PLoS One Abstract Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10-20% (show more...)Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10-20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: CD63/ uPA non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Characterization: Protein analysis Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ uPA ELISA Antibody details provided? No Detected EV-associated proteins uPA Characterization: Particle analysis None

	EV150017	1/1	Homo sapiens	NAY	(d)(U)C	Baglio SR	2015	33%
Study summary Full title Human bone marrow- and adipose-mesenchymal stem cells secrete exosomes enriched in distinctive miRNA and tRNA species All authors Baglio SR, Rooijers K, Koppers-Lalic D, Verweij FJ, Pérez Lanzón M, Zini N, Naaijkens B, Perut F, Niessen HW, Baldini N, Pegtel DM Journal Stem Cell Res Ther Abstract INTRODUCTION: Administration of mesenchymal stem cells (MSCs) represents a promising treatment optio (show more...)INTRODUCTION: Administration of mesenchymal stem cells (MSCs) represents a promising treatment option for patients suffering from immunological and degenerative disorders. Accumulating evidence indicates that the healing effects of MSCs are mainly related to unique paracrine properties, opening opportunities for secretome-based therapies. Apart from soluble factors, MSCs release functional small RNAs via extracellular vesicles (EVs) that seem to convey essential features of MSCs. Here we set out to characterize the full small RNAome of MSC-produced exosomes. METHODS: We set up a protocol for isolating exosomes released by early passage adipose- (ASC) and bone marrow-MSCs (BMSC) and characterized them via electron microscopy, protein analysis and small RNA-sequencing. We developed a bioinformatics pipeline to define the exosome-enclosed RNA species and performed the first complete small RNA characterization of BMSCs and ASCs and their corresponding exosomes in biological replicates. RESULTS: Our analysis revealed that primary ASCs and BMSCs have highly similar small RNA expression profiles dominated by miRNAs and snoRNAs (together 64-71 %), of which 150-200 miRNAs are present at physiological levels. In contrast, the miRNA pool in MSC exosomes is only 2-5 % of the total small RNAome and is dominated by a minor subset of miRNAs. Nevertheless, the miRNAs in exosomes do not merely reflect the cellular content and a defined set of miRNAs are overrepresented in exosomes compared to the cell of origin. Moreover, multiple highly expressed miRNAs are precluded from exosomal sorting, consistent with the notion that these miRNAs are involved in functional repression of RNA targets. While ASC and BMSC exosomes are similar in RNA class distribution and composition, we observed striking differences in the sorting of evolutionary conserved tRNA species that seems associated with the differentiation status of MSCs, as defined by Sox2, POU5F1A/B and Nanog expression. CONCLUSIONS: We demonstrate that primary MSCs release small RNAs via exosomes, which are increasingly implicated in intercellular communications. tRNAs species, and in particular tRNA halves, are preferentially released and their specific sorting into exosomes is related to MSC tissue origin and stemness. These findings may help to understand how MSCs impact neighboring or distant cells with possible consequences for their therapeutic usage. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Adj. k-factor 256 (pelleting) / 256 (washing) Protein markers EV: CD81/ CD63 non-EV: Cell organelle protein Proteomics no Show all info Study aim Omics Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type SW32 Pelleting: adjusted k-factor 256.0 Wash: Rotor Type SW32 Wash: adjusted k-factor 256.0 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Detected contaminants Cell organelle protein Characterization: Particle analysis EM EM-type transmission EM Image type Close-up

	EV140026	1/3	Mus musculus	Corneal fibroblasts	DG Filtration UF dUC	Han KY	2015	33%
Study summary Full title Evidence for the Involvement of MMP14 in MMP2 Processing and Recruitment in Exosomes of Corneal Fibroblasts. All authors Han KY, Dugas-Ford J, Seiki M, Chang JH, Azar DT Journal Invest Ophthalmol Vis Sci Abstract Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlying mechani (show more...)Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlying mechanisms are poorly understood. In this study, we investigated exosomal transport of MMP14 and its target, MMP2, from corneal fibroblasts to vascular endothelial cells as a possible mechanism governing MMP14 activity in corneal angiogenesis. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG Filtration UF dUC Protein markers EV: TSG101/ MMP14/ actin/ proMMP2/ MMP2/ ITGB1 non-EV: COX4/ MAPK Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells Corneal fibroblasts EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 120 Density gradient Type Continuous Lowest density fraction 5% Highest density fraction 40% Orientation Bottom-up Rotor type SW 40 Ti Speed (g) 100000 Duration (min) 1080 Filtration steps 0.45µm > x > 0.22µm, Ultra filtration Cut-off size (kDa) 100 Membrane type NS Other Name other separation method dUC Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins MMP14/ actin/ proMMP2/ MMP2/ ITGB1/ TSG101 Detected contaminants COX4/ MAPK Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210504	2/5	Macaca mulatta	Brain tissue	(d)(U)C DG	Yelamanchili SV	2015	29%
Study summary Full title MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease. All authors Yelamanchili SV, Lamberty BG, Rennard DA, Morsey BM, Hochfelder CG, Meays BM, Levy E, Fox HS Journal PLoS Pathog Abstract Recent studies have found that extracellular vesicles (EVs) play an important role in normal and dis (show more...)Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders. (hide) EV-METRIC 29% (29th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Brain tissue Sample origin Simian immunodeficiency virus Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Macaca mulatta Sample Type Brain tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 37 Wash: time (min) 60 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.25M Highest density fraction 2.0M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 2 Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 6 Fraction processing Centrifugation Pelleting: duration (min) 60 Pelleting: speed (g) 100000 Characterization: Protein analysis None Protein Concentration Method BCA Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210504	3/5	Macaca mulatta	Brain tissue	(d)(U)C DG	Yelamanchili SV	2015	29%
Study summary Full title MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease. All authors Yelamanchili SV, Lamberty BG, Rennard DA, Morsey BM, Hochfelder CG, Meays BM, Levy E, Fox HS Journal PLoS Pathog Abstract Recent studies have found that extracellular vesicles (EVs) play an important role in normal and dis (show more...)Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders. (hide) EV-METRIC 29% (29th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Brain tissue Sample origin Simian immunodeficiency virus encephalitis Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Macaca mulatta Sample Type Brain tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 37 Wash: time (min) 60 Wash: speed (g) 100000 Density gradient Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.25M Highest density fraction 2.0M Total gradient volume, incl. sample (mL) 12 Sample volume (mL) 2 Speed (g) 200000 Duration (min) 960 Fraction volume (mL) 6 Fraction processing Centrifugation Pelleting: duration (min) 60 Pelleting: speed (g) 100000 Characterization: Protein analysis None Protein Concentration Method BCA Characterization: RNA analysis RNA analysis Type (RT)(q)PCR/ RNAsequencing Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210032	1/2	Homo sapiens	Blood plasma	(d)(U)C	Linares, Romain	2015	29%
Study summary Full title High-speed centrifugation induces aggregation of extracellular vesicles All authors Romain Linares, Sisareuth Tan, Céline Gounou, Nicolas Arraud, Alain R Brisson Journal J Extracell Vesicles Abstract Plasma and other body fluids contain cell-derived extracellular vesicles (EVs), which participate in (show more...)Plasma and other body fluids contain cell-derived extracellular vesicles (EVs), which participate in physiopathological processes and have potential biomedical applications. In order to isolate, concentrate and purify EVs, high-speed centrifugation is often used. We show here, using electron microscopy, receptor-specific gold labelling and flow cytometry, that high-speed centrifugation induces the formation of EV aggregates composed of a mixture of EVs of various phenotypes and morphologies. The presence of aggregates made of EVs of different phenotypes may lead to erroneous interpretation concerning the existence of EVs harbouring surface antigens from different cell origins. (hide) EV-METRIC 29% (61st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Blood plasma Sample origin 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) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Protein markers EV: Annexin A5/ CD235a/ CD41 non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry Gallios (Beckman Coulter) Calibration bead size 0.4 Antibody details provided? No Detected EV-associated proteins Annexin A5/ CD235a/ CD41 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Immuno-EM/ Cryo-EM EM protein Annexin A5; CD235a; CD41 Image type Close-up, Wide-field EV concentration Yes

	EV150056	1/1	Homo sapiens Mus musculus	NAY	(d)(U)C Filtration	Takeda YS	2015	29%
Study summary Full title Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells All authors Takeda YS, Xu Q Journal PLoS One Abstract Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential (show more...)Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1 week. After the treatment with PC12-derived exosomes, MSCs developed neuron-like morphology, and gene and protein expressions of neuronal markers were upregulated. Microarray analysis showed that the expression of miR-125b, which is known to play a role in neuronal differentiation of stem cells, was much higher in PC12-derived exosomes than in exosomes from B16-F10 melanoma cells. These results suggest that the delivery of miRNAs contained in PC12-derived exosomes is a possible mechanism explaining the neuronal differentiation of MSC. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Adj. k-factor 156.9 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 150 Pelleting: rotor type 70Ti Pelleting: adjusted k-factor 156.9 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis DLS EM EM-type transmission EM Image type Wide-field

	EV150050	1/1	Bos bovis	Milk	(d)(U)C DG Filtration	Sun J	2015	29%
Study summary Full title MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection All authors Sun J, Aswath K, Schroeder SG, Lippolis JD, Reinhardt TA, Sonstegard TS Journal BMC Genomics Abstract BACKGROUND: Milk exosomes are a rich source of microRNAs (miRNAs) that are protected from degradatio (show more...)BACKGROUND: Milk exosomes are a rich source of microRNAs (miRNAs) that are protected from degradation. Ingestion of milk and subsequent absorption of miRNAs into recipient cells by endocytosis may play a role in the regulation of neonatal innate and adaptive immunity. In contrast, the miRNA content of milk exosomes may also be indicative of a lactating animal's health; whereby, the presence or absence of specific miRNAs could serve as biomarkers for early detection of bacterial infection that can lead to mastitis. In the present study, we therefore analyzed and compared miRNA expression profiles of milk exosomes from four Holstein cows obtained during mid-lactation prior to and after infection (48 h) of the mammary gland with Staphylococcus aureus. METHODS: Milk exosomes, purified from control and S. aureus infected cows, were extracted for RNA. Following preparation indexed libraries from both groups the samples were subjected to next generation sequencing. RESULTS: Next generation sequencing of eight, unpooled small RNA libraries derived from milk exosomes produced about 60.5 million high-quality, bovine-specific sequence reads for comparison of miRNA expression between treatments. Sequence identity analysis showed the miRNAs make up about 13 % of the average RNA content of these exosomes. Although 417 known bovine miRNAs were identified, miRNAs represented the least diverse class of RNA accounting for only 1 % of all unique sequences. The 20 most prevalent unique sequences within this class accounted for about 90 % of the total miRNA-associated reads across samples. Non-annotated, unique reads provided evidence for another 303 previously unknown bovine miRNAs. Expression analyses found 14 known bovine microRNAs significantly differed in frequency between exosomes from infected and control animals. CONCLUSIONS: Our survey of miRNA expression from uninfected milk exosomes and those produced in response to infection provides new and comprehensive information supporting a role for delivery into milk of specific miRNAs involved in immune response. In particular, bta-miR-142-5p, and -223 are potential biomarkers for early detection of bacterial infection of the mammary gland. Additionally, 22 mammary-expressed genes involved in regulation of host immune processes and response to inflammation were identified as potential binding targets of the differentially expressed miRNAs. (hide) EV-METRIC 29% (41st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 157.1 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Biomarker Sample Species Bos bovis Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type 50.2Ti Pelleting: adjusted k-factor 157.1 Density gradient Lowest density fraction 5 Highest density fraction 43 Orientation Bottom-up Speed (g) 200000 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis None

	EV150016	1/2	Homo sapiens	NAY	(d)(U)C DC Filtration	Pospichalova V	2015	29%
Study summary Full title Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer All authors Pospichalova V, Svoboda J, Dave Z, Kotrbova A, Kaiser K, Klemova D, Ilkovics L, Hampl A, Crha I, Jandakova E, Minar L, Weinberger V, Bryja V Journal J Extracell Vesicles Abstract Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and quali (show more...)Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes / microvesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration Adj. k-factor 253.9 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Technical Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 190 Pelleting: rotor type SW28 Pelleting: adjusted k-factor 253.9 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

	EV150060	3/4	Homo sapiens	Semen	(d)(U)C DG Filtration	Madison MN	2015	29%
Study summary Full title Exosomes in human semen restrict HIV-1 transmission by vaginal cells and block intravaginal replication of LP-BM5 murine AIDS virus complex All authors Madison MN, Jones PH, Okeoma CM Journal Virology Abstract Exosomes are membranous extracellular nanovesicles secreted by diverse cell types. Exosomes from hea (show more...)Exosomes are membranous extracellular nanovesicles secreted by diverse cell types. Exosomes from healthy human semen have been shown to inhibit HIV-1 replication and to impair progeny virus infectivity. In this study, we examined the ability of healthy human semen exosomes to restrict HIV-1 and LP-BM5 murine AIDS virus transmission in three different model systems. We show that vaginal cells internalize exosomes with concomitant transfer of functional mRNA. Semen exosomes blocked the spread of HIV-1 from vaginal epithelial cells to target cells in our cell-to-cell infection model and suppressed transmission of HIV-1 across the vaginal epithelial barrier in our trans-well model. Our in vivo model shows that human semen exosomes restrict intravaginal transmission and propagation of murine AIDS virus. Our study highlights an antiretroviral role for semen exosomes that may be harnessed for the development of novel therapeutic strategies to combat HIV-1 transmission. (hide) EV-METRIC 29% (56th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Semen Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 255.8 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Semen Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Density gradient Lowest density fraction 2.5 Highest density fraction 25 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Particle analysis None

	EV150060	4/4	Homo sapiens	Serum	(d)(U)C DG Filtration	Madison MN	2015	29%
Study summary Full title Exosomes in human semen restrict HIV-1 transmission by vaginal cells and block intravaginal replication of LP-BM5 murine AIDS virus complex All authors Madison MN, Jones PH, Okeoma CM Journal Virology Abstract Exosomes are membranous extracellular nanovesicles secreted by diverse cell types. Exosomes from hea (show more...)Exosomes are membranous extracellular nanovesicles secreted by diverse cell types. Exosomes from healthy human semen have been shown to inhibit HIV-1 replication and to impair progeny virus infectivity. In this study, we examined the ability of healthy human semen exosomes to restrict HIV-1 and LP-BM5 murine AIDS virus transmission in three different model systems. We show that vaginal cells internalize exosomes with concomitant transfer of functional mRNA. Semen exosomes blocked the spread of HIV-1 from vaginal epithelial cells to target cells in our cell-to-cell infection model and suppressed transmission of HIV-1 across the vaginal epithelial barrier in our trans-well model. Our in vivo model shows that human semen exosomes restrict intravaginal transmission and propagation of murine AIDS virus. Our study highlights an antiretroviral role for semen exosomes that may be harnessed for the development of novel therapeutic strategies to combat HIV-1 transmission. (hide) EV-METRIC 29% (72nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Adj. k-factor 255.8 (pelleting) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 30 Pelleting: rotor type SW41 Pelleting: adjusted k-factor 255.8 Density gradient Lowest density fraction 2.5 Highest density fraction 25 Orientation Bottom-up Speed (g) 100000 Filtration steps 0.45µm > x > 0.22µm, Characterization: Particle analysis None

	EV150064	3/3	Homo sapiens	NAY	(d)(U)C DG Filtration	de Vrij J	2015	29%
Study summary Full title Glioblastoma-derived extracellular vesicles modify the phenotype of monocytic cells All authors de Vrij J, Maas SL, Kwappenberg KM, Schnoor R, Kleijn A, Dekker L, Luider TM, de Witte LD, Litjens M, van Strien ME, Hol EM, Kroonen J, Robe PA, Lamfers ML, Schilham MW, Broekman ML Journal Int J Cancer Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor and is without exception lethal (show more...)Glioblastoma multiforme (GBM) is the most common primary brain tumor and is without exception lethal. GBMs modify the immune system, which contributes to the aggressive nature of the disease. Particularly, cells of the monocytic lineage, including monocytes, macrophages and microglia, are affected. We investigated the influence of GBM-derived extracellular vesicles (EVs) on the phenotype of monocytic cells. Proteomic profiling showed GBM EVs to be enriched with proteins functioning in extracellular matrix interaction and leukocyte migration. GBM EVs appeared to skew the differentiation of peripheral blood-derived monocytes to alternatively activated/M2-type macrophages. This was observed for EVs from an established cell line, as well as for EVs from primary cultures of GBM stem-like cells (GSCs). Unlike EVs of non-GBM origin, GBM EVs induced modified expression of cell surface proteins, modified cytokine secretion (e.g., an increase in vascular endothelial growth factor and IL-6) and increased phagocytic capacity of the macrophages. Most pronounced effects were observed upon incubation with EVs from mesenchymal GSCs. GSC EVs also affected primary human microglia, resulting in increased expression of Membrane type 1-matrix metalloproteinase, a marker for GBM microglia and functioning as tumor-supportive factor. In conclusion, GBM-derived EVs can modify cells of the monocytic lineage, which acquire characteristics that resemble the tumor-supportive phenotypes observed in patients. (hide) EV-METRIC 29% (68th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Filtration Protein markers EV: non-EV: Proteomics yes Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Characterization: Particle analysis TRPS

	EV150035	1/1	Bos bovis	Milk	(d)(U)C DC Filtration	Arntz OJ	2015	29%
Study summary Full title Oral administration of bovine milk derived extracellular vesicles attenuates arthritis in two mouse models All authors Arntz OJ, Pieters BC, Oliveira MC, Broeren MG, Bennink MB, de Vries M, van Lent PL, Koenders MI, van den Berg WB, van der Kraan PM, van de Loo FA Journal Mol Nutr Food Res Abstract SCOPE: This study shows the effect of bovine milk derived extracellular vesicles (BMEVs) on spontane (show more...)SCOPE: This study shows the effect of bovine milk derived extracellular vesicles (BMEVs) on spontaneous polyarthritis in IL-1Ra-deficient mice and collagen-induced arthritis. METHODS AND RESULTS: BMEVs were isolated from semi-skimmed milk by ultracentrifugation and the particle size was around 100 nm by dynamic light scattering and electron microscopy. BMEVs expressed exosome marker CD63, immunoregulatory microRNA's (miR-30a, -223, -92a), and milk-specific beta-casein and beta-lactoglobulin mRNA. In vitro, PKH-67-labeled BMEVs were taken up by RAW264.7, splenocytes, and intestinal cells as determined by flow cytometry and confocal microscopy. IL-1Ra(-/-) mice received BMEVs by daily oral gavage starting at wk 5 till 15 after birth and collagen-induced arthritis mice via their drinking water starting 1 wk before immunization till day 40. Macroscopically, BMEV treatment delayed the onset of arthritis and histology showed diminished cartilage pathology and bone marrow inflammation in both models. BMEV treatment also reduced the serum levels of MCP-1 and IL-6 and their production by splenic cells. BMEV treatment diminished the anticollagen IgG2a levels, which was accompanied by reduced splenic Th1 (Tbet) and Th17 (ROR?T) mRNA. CONCLUSION: This is the first report that oral delivery of BMEVs ameliorates experimental arthritis and this warrants further research to determine whether this beneficial effect can be seen in rheumatoid arthritis patients. (hide) EV-METRIC 29% (41st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Milk Sample origin NAY Focus vesicles extracellular vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DC Filtration Protein markers EV: non-EV: Proteomics yes Show all info Study aim Function Sample Species Bos bovis Sample Type Milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Characterization: Particle analysis DLS EM EM-type immune EM EM protein CD63 Image type Close-up

	EV220077	1/2	Homo sapiens	Serum	(d)(U)C ExoQuick	Zaharie F	2015	25%
Study summary Full title Exosome-Carried microRNA-375 Inhibits Cell Progression and Dissemination via Bcl-2 Blocking in Colon Cancer. All authors Zaharie F, Muresan MS, Petrushev B, Berce C, Gafencu GA, Selicean S, Jurj A, Cojocneanu-Petric R, Lisencu CI, Pop LA, Pileczki V, Eniu D, Muresan MA, Zaharie R, Berindan-Neagoe I, Tomuleasa C, Irimie A Journal J Gastrointestin Liver Dis Abstract Worldwide, colorectal cancer (CRC) is the third most common cancer in men and second in women. The a (show more...)Worldwide, colorectal cancer (CRC) is the third most common cancer in men and second in women. The aim of the current study was to identify whether the miR-375 is indeed down-regulated in metastatic CRC and if it could be considered as a potential minimally invasive prognostic biomarker for CRC. (hide) EV-METRIC 25% (67th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin 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) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: TSG101/ CD81/ beta actin non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins TSG101/ CD81/ beta-actin Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220077	2/2	Homo sapiens	Serum	(d)(U)C ExoQuick	Zaharie F	2015	25%
Study summary Full title Exosome-Carried microRNA-375 Inhibits Cell Progression and Dissemination via Bcl-2 Blocking in Colon Cancer. All authors Zaharie F, Muresan MS, Petrushev B, Berce C, Gafencu GA, Selicean S, Jurj A, Cojocneanu-Petric R, Lisencu CI, Pop LA, Pileczki V, Eniu D, Muresan MA, Zaharie R, Berindan-Neagoe I, Tomuleasa C, Irimie A Journal J Gastrointestin Liver Dis Abstract Worldwide, colorectal cancer (CRC) is the third most common cancer in men and second in women. The a (show more...)Worldwide, colorectal cancer (CRC) is the third most common cancer in men and second in women. The aim of the current study was to identify whether the miR-375 is indeed down-regulated in metastatic CRC and if it could be considered as a potential minimally invasive prognostic biomarker for CRC. (hide) EV-METRIC 25% (67th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Serum Sample origin CCR patients with liver metastases Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: TSG101/ CD81/ beta actin non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins TSG101/ CD81/ beta-actin Characterization: Lipid analysis No EM EM-type Scanning-EM Image type Wide-field

	EV220043	1/4	Homo sapiens	U-87MG	(d)(U)C Other/ Total Exosome RNA and Protein Isolation Kit (Invitrogen)	Yang T	2015	25%
Study summary Full title Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. All authors Yang T, Martin P, Fogarty B, Brown A, Schurman K, Phipps R, Yin VP, Lockman P, Bai S Journal Pharm Res Abstract The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. (show more...)The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. (hide) EV-METRIC 25% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells U-87MG EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Other/ Total Exosome RNA and Protein Isolation Kit (Invitrogen) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Not detected EV-associated proteins CD81/ CD63/ CD9 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported EM EM-type Scanning-EM Image type Close-up, Wide-field

	EV220043	2/4	Homo sapiens	bEND.3	(d)(U)C Other/ Total Exosome RNA and Protein Isolation Kit (Invitrogen)	Yang T	2015	25%
Study summary Full title Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. All authors Yang T, Martin P, Fogarty B, Brown A, Schurman K, Phipps R, Yin VP, Lockman P, Bai S Journal Pharm Res Abstract The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. (show more...)The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. (hide) EV-METRIC 25% (64th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Commercial method Protein markers EV: CD81/ CD63/ CD9 non-EV: None Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells bEND.3 EV-harvesting Medium EV-depleted medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Other/ Total Exosome RNA and Protein Isolation Kit (Invitrogen) Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63 Not detected EV-associated proteins CD81/ CD9 ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ CD9 Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Not Reported EM EM-type Scanning-EM Image type Close-up, Wide-field

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