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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200168	1/5	Homo sapiens	Rh36	(d)(U)C	Ghayad, Sandra E	2016	33%
Study summary Full title Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts All authors Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab Journal Sci Rep Abstract Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (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 (Differential) (ultra)centrifugation Protein markers EV: TSG101/ GAPDH/ HSC70 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Rh36 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins HSC70/ GAPDH/ TSG101 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Capillary electrophoresis (e.g. Bioanalyzer);Microarray Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.2 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Scanning-EM Image type Wide-field Report size (nm) 40-120

	EV200168	2/5	Homo sapiens	Rh30	(d)(U)C	Ghayad, Sandra E	2016	33%
Study summary Full title Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts All authors Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab Journal Sci Rep Abstract Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (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 (Differential) (ultra)centrifugation Protein markers EV: TSG101/ Pax3-FOXO1/ GAPDH/ HSC70 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Rh30 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins HSC70/ GAPDH/ Pax3-FOXO1/ TSG101 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.2 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Scanning-EM Image type Wide-field Report size (nm) 40-120

	EV200168	3/5	Homo sapiens	RD	(d)(U)C	Ghayad, Sandra E	2016	33%
Study summary Full title Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts All authors Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab Journal Sci Rep Abstract Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (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 (Differential) (ultra)centrifugation Protein markers EV: TSG101/ GAPDH/ HSC70 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells RD EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins TSG101/ HSC70/ GAPDH Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.2 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Scanning-EM Image type Wide-field Report size (nm) 40-120

	EV200168	4/5	Homo sapiens	Rh41	(d)(U)C	Ghayad, Sandra E	2016	33%
Study summary Full title Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts All authors Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab Journal Sci Rep Abstract Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (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 (Differential) (ultra)centrifugation Protein markers EV: TSG101/ Pax3-FOXO1/ GAPDH/ HSC70 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Rh41 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins HSC70/ GAPDH/ Pax3-FOXO1/ TSG101 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.2 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Scanning-EM Image type Wide-field Report size (nm) 40-120

	EV200168	5/5	Homo sapiens	JR1	(d)(U)C	Ghayad, Sandra E	2016	33%
Study summary Full title Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts All authors Sandra E Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Rihab Nasr, Mona Diab-Assaf, Claude Chelala, Raya Saab Journal Sci Rep Abstract Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX (show more...)Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. (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 (Differential) (ultra)centrifugation Protein markers EV: TSG101/ GAPDH/ HSC70 non-EV: Calnexin Proteomics no Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells JR1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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) 100000 Wash: volume per pellet (ml) Not specified Wash: time (min) 70 Wash: Rotor Type Not specified Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Lysis buffer provided? Yes Detected EV-associated proteins HSC70/ GAPDH/ TSG101 Not detected contaminants Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;Microarray Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.2 Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Scanning-EM Image type Wide-field Report size (nm) 40-120

	EV160012	1/8	Homo sapiens	MCF7	(d)(U)C Filtration	Hannafon BN	2016	33%
Study summary Full title Plasma exosome microRNAs are indicative of breast cancer. All authors Hannafon BN, Trigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, Zhao ZJ, Blick KE, Dooley WC, Ding WQ. Journal Breast Cancer Res Treat Abstract BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific (show more...)BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. (hide) EV-METRIC 33% (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 Filtration Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Preparation of EDS 2h at 100,000g 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 Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 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 Mean Reported size (nm) 103.3 EV concentration Yes EM EM-type Immuno-EM EM protein CD63 Image type Close-up, Wide-field

	EV160012	2/8	Homo sapiens	MDAMB231	(d)(U)C Filtration	Hannafon BN	2016	33%
Study summary Full title Plasma exosome microRNAs are indicative of breast cancer. All authors Hannafon BN, Trigoso YD, Calloway CL, Zhao YD, Lum DH, Welm AL, Zhao ZJ, Blick KE, Dooley WC, Ding WQ. Journal Breast Cancer Res Treat Abstract BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific (show more...)BACKGROUND: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring. METHODS: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis. RESULTS: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels. CONCLUSIONS: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer. (hide) EV-METRIC 33% (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 Filtration Protein markers EV: CD63 non-EV: Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MDAMB231 EV-harvesting Medium EV-depleted medium Preparation of EDS 2h at 100,000g 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 Not specified Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 10 Wash: time (min) 60 Wash: Rotor Type Not specified Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 99.6 EV concentration Yes EM EM-type Immuno-EM EM protein CD63 Image type Close-up, Wide-field

	EV160011	1/4	Homo sapiens	BCBL1	(d)(U)C ExoQuick Filtration	Hoshina S	2016	33%
Study summary Full title Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines. All authors Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H. Journal PLoS One Abstract Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide) EV-METRIC 33% (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 Kaposi sarcoma-associated herpesvirus-infected Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick Filtration Protein markers EV: Lyn/ CD63/ HSP70 non-EV: KSHV ORF45 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells BCBL1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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 SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins CD63/ Lyn/ HSP70 Detected contaminants KSHV ORF45 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNA sequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV160011	2/4	Homo sapiens	TY1	(d)(U)C ExoQuick Filtration	Hoshina S	2016	33%
Study summary Full title Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines. All authors Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H. Journal PLoS One Abstract Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide) EV-METRIC 33% (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 Kaposi sarcoma-associated herpesvirus-infected Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick Filtration Protein markers EV: HSP70/ Lyn/ CD63 non-EV: KSHV ORF45 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells TY1 EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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 SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins CD63/ Lyn/ HSP70 Not detected contaminants KSHV ORF45 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV160011	3/4	Homo sapiens	LCL	(d)(U)C ExoQuick Filtration	Hoshina S	2016	33%
Study summary Full title Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines. All authors Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H. Journal PLoS One Abstract Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide) EV-METRIC 33% (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 Epstein-Barr virus-infected Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C ExoQuick Filtration Protein markers EV: HSP70/ Lyn/ CD63 non-EV: KSHV ORF45 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells LCL EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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 SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins CD63/ HSP70/ Lyn Not detected contaminants KSHV ORF45 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV160011	4/4	Homo sapiens	Bjab	(d)(U)C ExoQuick Filtration	Hoshina S	2016	33%
Study summary Full title Profile of Exosomal and Intracellular microRNA in Gamma-Herpesvirus-Infected Lymphoma Cell Lines. All authors Hoshina S, Sekizuka T, Kataoka M, Hasegawa H, Hamada H, Kuroda M, Katano H. Journal PLoS One Abstract Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorte (show more...)Exosomes are small vesicles released from cells, into which microRNAs (miRNA) are specifically sorted and accumulated. Two gamma-herpesviruses, Kaposi sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), encode miRNAs in their genomes and express virus-encoded miRNAs in cells and exosomes. However, there is little information about the detailed distribution of virus-encoded miRNAs in cells and exosomes. In this study, we thus identified virus- and host-encoded miRNAs in exosomes released from KSHV- or EBV-infected lymphoma cell lines and compared them with intracellular miRNAs using a next-generation sequencer. Sequencing analysis demonstrated that 48% of the annotated miRNAs in the exosomes from KSHV-infected cells originated from KSHV. Human mir-10b-5p and mir-143-3p were much more highly concentrated in exosomes than in cells. Exosomes contained more nonexact mature miRNAs that did not exactly match those in miRBase than cells. Among the KSHV-encoded miRNAs, miRK12-3-5p was the most abundant exact mature miRNA in both cells and exosomes that exactly matched those in miRBase. Recently identified EXOmotifs, nucleotide motifs that control the loading of miRNAs into exosomes were frequently found within the sequences of KSHV-encoded miRNAs, and the presence of the EXOmotif CCCT or CCCG was associated with the localization of miRNA in exosomes in KSHV-infected cells. These observations suggest that specific virus-encoded miRNAs are sorted by EXOmotifs and accumulate in exosomes in virus-infected cells. (hide) EV-METRIC 33% (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 ExoQuick Filtration Protein markers EV: HSP70/ Lyn/ CD63 non-EV: KSHV ORF45 Proteomics no Show all info Study aim Biogenesis/cargo sorting/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Bjab EV-harvesting Medium EV-depleted medium Preparation of EDS overnight (16h) 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 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 SW 32 Ti Pelleting: speed (g) 110000 Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Detected EV-associated proteins Lyn/ CD63/ HSP70 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR;RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Wide-field

	EV160008	3/3	Rattus norvegicus	INS1	(d)(U)C	Cianciaruso C	2016	33%
Study summary Full title Primary Human and Rat β-Cells Release the Intracellular Autoantigens GAD65, IA-2, and Proinsulin in Exosomes Together With Cytokine-Induced Enhancers of Immunity. All authors Cianciaruso C, Phelps EA, Pasquier M, Hamelin R, Demurtas D, Alibashe Ahmed M, Piemonti L, Hirosue S, Swartz MA, De Palma M, Hubbell JA, Baekkeskov S Journal Diabetes Abstract The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1 (show more...)The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2, and proinsulin in exosomes, which are taken up by and activate dendritic cells. Accordingly, the anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T-cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Cytokine-induced endoplasmic reticulum stress enhanced exosome secretion by β-cells; induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96, and ORP150; and increased exosomal stimulation of antigen-presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in the initiation of autoimmune responses in T1D. (hide) EV-METRIC 33% (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 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 (d)(U)C Protein markers EV: Alix non-EV: None Proteomics yes Show all info Study aim Function, Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells INS1 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA 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: speed (g) 110000 Wash: time (min) 70 Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins Alix Proteomics database No Characterization: Lipid analysis No

	EV220065	1/2	Bos taurus	Blood plasma	DG (d)(U)C Filtration	Crookenden MA	2016	29%
Study summary Full title Short communication: Proteins from circulating exosomes represent metabolic state in transition dairy cows. All authors Crookenden MA, Walker CG, Peiris H, Koh Y, Heiser A, Loor JJ, Moyes KM, Murray A, Dukkipati VSR, Kay JK, Meier S, Roche JR, Mitchell MD Journal J Dairy Sci Abstract Biomarkers that identify prepathological disease could enhance preventive management, improve animal (show more...)Biomarkers that identify prepathological disease could enhance preventive management, improve animal health and productivity, and reduce costs. Circulating extracellular vesicles, particularly exosomes, are considered to be long-distance, intercellular communication systems in human medicine. Exosomes provide tissue-specific messages of functional state and can alter the cellular activity of recipient tissues through their protein and microRNA content. We hypothesized that exosomes circulating in the blood of cows during early lactation would contain proteins representative of the metabolic state of important tissues, such as liver, which play integral roles in regulating the physiology of cows postpartum. From a total of 150 cows of known metabolic phenotype, 10 cows were selected with high (n=5/ high risk) and low (n=5/ low risk) concentrations of nonesterified fatty acids, β-hydroxybutyrate, and liver triacylglycerol during wk 1 and 2 after calving. Exosomes were extracted from blood on the day of calving (d 0) and postcalving at wk 1 and wk 4, and their protein composition was determined by mass spectroscopy. Extracellular vesicle protein concentration and the number of exosome vesicles were not affected by risk category/ however, the exosome protein cargo differed between the groups, with proteins at each time point identified as being unique to the high- and low-risk groups. The proteins α-2 macroglobulin, fibrinogen, and oncoprotein-induced transcript 3 were unique to the high-risk cows on d 0 and have been associated with metabolic syndrome and liver function in humans. Their presence may indicate a more severe inflammatory state and a greater degree of liver dysfunction in the high-risk cows than in the low-risk cows, consistent with the high-risk cows' greater plasma β-hydroxybutyrate and liver triacylglycerol concentrations. The commonly shared proteins and those unique to the low-risk category indicate a role for exosomes in immune function. The data provide preliminary evidence of a potential role for exosomes in the immune function in transition dairy cows and exosomal protein cargo as biomarkers of metabolic state. (hide) EV-METRIC 29% (60th 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 Low risk metabolic disease 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Blood plasma 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: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 75 Wash: speed (g) 100000 Density gradient Type Discontinuous Sample volume (mL) 0.5 Orientation Top-down Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other/ DC protein assay Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample

	EV220065	2/2	Bos taurus	Blood plasma	DG (d)(U)C Filtration	Crookenden MA	2016	29%
Study summary Full title Short communication: Proteins from circulating exosomes represent metabolic state in transition dairy cows. All authors Crookenden MA, Walker CG, Peiris H, Koh Y, Heiser A, Loor JJ, Moyes KM, Murray A, Dukkipati VSR, Kay JK, Meier S, Roche JR, Mitchell MD Journal J Dairy Sci Abstract Biomarkers that identify prepathological disease could enhance preventive management, improve animal (show more...)Biomarkers that identify prepathological disease could enhance preventive management, improve animal health and productivity, and reduce costs. Circulating extracellular vesicles, particularly exosomes, are considered to be long-distance, intercellular communication systems in human medicine. Exosomes provide tissue-specific messages of functional state and can alter the cellular activity of recipient tissues through their protein and microRNA content. We hypothesized that exosomes circulating in the blood of cows during early lactation would contain proteins representative of the metabolic state of important tissues, such as liver, which play integral roles in regulating the physiology of cows postpartum. From a total of 150 cows of known metabolic phenotype, 10 cows were selected with high (n=5/ high risk) and low (n=5/ low risk) concentrations of nonesterified fatty acids, β-hydroxybutyrate, and liver triacylglycerol during wk 1 and 2 after calving. Exosomes were extracted from blood on the day of calving (d 0) and postcalving at wk 1 and wk 4, and their protein composition was determined by mass spectroscopy. Extracellular vesicle protein concentration and the number of exosome vesicles were not affected by risk category/ however, the exosome protein cargo differed between the groups, with proteins at each time point identified as being unique to the high- and low-risk groups. The proteins α-2 macroglobulin, fibrinogen, and oncoprotein-induced transcript 3 were unique to the high-risk cows on d 0 and have been associated with metabolic syndrome and liver function in humans. Their presence may indicate a more severe inflammatory state and a greater degree of liver dysfunction in the high-risk cows than in the low-risk cows, consistent with the high-risk cows' greater plasma β-hydroxybutyrate and liver triacylglycerol concentrations. The commonly shared proteins and those unique to the low-risk category indicate a role for exosomes in immune function. The data provide preliminary evidence of a potential role for exosomes in the immune function in transition dairy cows and exosomal protein cargo as biomarkers of metabolic state. (hide) EV-METRIC 29% (60th 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 High risk metabolic disease 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 Density gradient (Differential) (ultra)centrifugation Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Bos taurus Sample Type Blood plasma 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: speed (g) 100000 Wash: volume per pellet (ml) 30 Wash: time (min) 75 Wash: speed (g) 100000 Density gradient Type Discontinuous Sample volume (mL) 0.5 Orientation Top-down Fraction processing Centrifugation Pelleting: duration (min) 120 Pelleting: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Other/ DC protein assay Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 3.74e+10

	EV220045	3/4	Homo sapiens	Blood plasma	IAF	Zhao Z	2016	29%
Study summary Full title A microfluidic ExoSearch chip for multiplexed exosome detection towards blood-based ovarian cancer diagnosis. All authors Zhao Z, Yang Y, Zeng Y, He M Journal Lab Chip Abstract Tumor-derived circulating exosomes, enriched with a group of tumor antigens, have been recognized as (show more...)Tumor-derived circulating exosomes, enriched with a group of tumor antigens, have been recognized as a promising biomarker source for cancer diagnosis via a less invasive procedure. Quantitatively pinpointing exosome tumor markers is appealing, yet challenging. In this study, we developed a simple microfluidic approach (ExoSearch) which provides enriched preparation of blood plasma exosomes for in situ, multiplexed detection using immunomagnetic beads. The ExoSearch chip offers a robust, continuous-flow design for quantitative isolation and release of blood plasma exosomes in a wide range of preparation volumes (10 μL to 10 mL). We employed the ExoSearch chip for blood-based diagnosis of ovarian cancer by multiplexed measurement of three exosomal tumor markers (CA-125, EpCAM, CD24) using a training set of ovarian cancer patient plasma, which showed significant diagnostic power (a.u.c. = 1.0, p = 0.001) and was comparable with the standard Bradford assay. This work provides an essentially needed platform for utilization of exosomes in clinical cancer diagnosis, as well as fundamental exosome research. (hide) EV-METRIC 29% (60th 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 Ovarian cancer 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 Immunoaffinity capture (non-commercial) Protein markers EV: CD24/ EpCAM/ CA-125 non-EV: None Proteomics no Show all info Study aim New methodological development Sample Species Homo sapiens Sample Type Blood plasma Separation Method Immunoaffinity capture Selected surface protein(s) CD24/ CA-125/ EpCAM Characterization: Protein analysis Protein Concentration Method Bradford Fluorescent NTA Antibody details provided? Yes Detected EV-associated proteins CD24/ EpCAM/ CA-125 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV220015	6/9	Homo sapiens	Serum	(d)(U)C	Verma VK	2016	29%
Study summary Full title Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles. All authors Verma VK, Li H, Wang R, Hirsova P, Mushref M, Liu Y, Cao S, Contreras PC, Malhi H, Kamath PS, Gores GJ, Shah VH Journal J Hepatol Abstract The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macropha (show more...)The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. (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 Alcohol hepatitis 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: CD40/ IFN-gamma/ IL-23/ IL1-Ra/ PAI-1/ MIF/ IL-16/ MIP-1alpha/ GROalpha/ C5/5a/ I-309/ GM-CSF/ G-CSF/ sICAM-1/ IL-17e/ TNF-alpha/ I-TAC/ IL-13/ RANTES/ IL1-alpha/ sTREM-1/ MCP1/ IL-2/ IL-4/ MIP-1beta/ IL-27/ IL-17/ IL-12p70/ IL-6/ IL-1beta/ IP-10/ IL-5/ SDF-1/ IL-10/ 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 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: rotor type SW 32 Ti Pelleting: speed (g) 100000 Wash: time (min) 120 Wash: Rotor Type SW 32 Ti Wash: speed (g) 110000 Characterization: Protein analysis Protein Concentration Method Not determined Detected EV-associated proteins CD40/ IFN-gamma/ IL-23/ IL1-Ra/ PAI-1/ MIF/ IL-16/ MIP-1alpha/ GROalpha/ C5/5a/ I-309/ GM-CSF/ G-CSF/ sICAM-1/ IL-17e/ TNF-alpha/ I-TAC/ IL-13/ RANTES/ IL1-alpha/ sTREM-1/ MCP1/ Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes

	EV210443	2/2	Homo sapiens	CSF	(d)(U)C Filtration	Akers JC	2016	29%
Study summary Full title Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF). All authors Akers JC, Ramakrishnan V, Nolan JP, Duggan E, Fu CC, Hochberg FH, Chen CC, Carter BS Journal PLoS One Abstract Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patient (show more...)Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs <150 nm in diameter, NTA detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2-3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations. (hide) EV-METRIC 29% (60th 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 CSF Sample origin Glioblastoma 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 Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type CSF 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: rotor type Type 70 Ti Pelleting: speed (g) 120000 Filtration steps > 0.45 µm, Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes TRPS Report type Not Reported EV concentration Yes Particle analysis: flow cytometry Flow cytometer type Custom build flow cytometer Hardware adjustment Stoner SA, Duggan E, Condello D, Guerrero A, Turk JR, Narayanan PK, et al. High sensitivity flow cytometry of membrane vesicles. Cytometry A. 2015. doi: 10.1002/cyto.a.22787 PMID: 26484737 Calibration bead size 0.12 Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Wide-field EV concentration Yes

	EV210402	1/1	Homo sapiens	Follicular fluid	(d)(U)C	Franz C	2016	29%
Study summary Full title Extracellular vesicles in human follicular fluid do not promote coagulation. All authors Franz C, Böing AN, Montag M, Strowitzki T, Markert UR, Mastenbroek S, Nieuwland R, Toth B Journal Reprod Biomed Online Abstract Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an (show more...)Body fluids contain extracellular vesicles expressing tissue factor on their surface and serve as an additional trigger for coagulation. During the menstrual cycle ovarian tissue restoration is mandatory and it is unknown whether follicular fluid might provide procoagulant substances. Within an observational study, follicular fluid from women undergoing IVF/intracytoplasmic sperm injection (ICSI) was analysed by fluorescence-activated cell sorting (FACS), electron microscopy, resistive pulse sensing (RPS), nanoparticle-tracking analysis (NTA) and fibrin generation tests (FGT). The presence of extracellular vesicles, especially CD9-positive extracellular vesicles in follicular fluid, was proven. However, clotting tests revealed no procoagulant properties of the detected extracellular vesicles. (hide) EV-METRIC 29% (62nd percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 Follicular fluid Sample origin Gonadotrophin-releasing hormone agonist 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: HSP70/ CD14/ EGFR/ CD24/ CD45/ L1CAM/ CD9 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Follicular fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: speed (g) 18890 Wash: volume per pellet (ml) 0.225 Wash: time (min) 30 Wash: speed (g) 18890 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry FACS Calibur (Becton Dickinson) Antibody details provided? No Detected EV-associated proteins HSP70/ CD9/ CD14/ EGFR/ CD24/ CD45/ L1CAM Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 70-1075 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 2.30e+10 TRPS Report type Size range/distribution Reported size (nm) 165-200 EV concentration Yes Particle yield as number of particles per milliliter of starting sample: 1.20e+8 EM EM-type Transmission-EM Image type Close-up

	EV210106	1/5	Homo sapiens	J82	(d)(U)C Filtration	Andreu, Zoraida	2016	29%
Study summary Full title Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression All authors Zoraida Andreu, Renan Otta Oshiro, Alberto Redruello, Soraya López-Martín, Cristina Gutiérrez-Vázquez, Esperanza Morato, Ana Isabel Marina, Carlos Olivier Gómez, María Yáñez-Mó Journal Eur J Pharm Sci. Abstract Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Cu (show more...)Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy. (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 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 (d)(U)C Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells J82 EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type JS-24.38 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 33 Wash: time (min) 60 Wash: Rotor Type JS-24.38 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210106	2/5	Homo sapiens	UMUC-3	(d)(U)C Filtration	Andreu, Zoraida	2016	29%
Study summary Full title Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression All authors Zoraida Andreu, Renan Otta Oshiro, Alberto Redruello, Soraya López-Martín, Cristina Gutiérrez-Vázquez, Esperanza Morato, Ana Isabel Marina, Carlos Olivier Gómez, María Yáñez-Mó Journal Eur J Pharm Sci. Abstract Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Cu (show more...)Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy. (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 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 (d)(U)C Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells UMUC-3 EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type JS-24.38 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 33 Wash: time (min) 60 Wash: Rotor Type JS-24.38 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210106	3/5	Homo sapiens	SW780	(d)(U)C Filtration	Andreu, Zoraida	2016	29%
Study summary Full title Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression All authors Zoraida Andreu, Renan Otta Oshiro, Alberto Redruello, Soraya López-Martín, Cristina Gutiérrez-Vázquez, Esperanza Morato, Ana Isabel Marina, Carlos Olivier Gómez, María Yáñez-Mó Journal Eur J Pharm Sci. Abstract Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Cu (show more...)Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high-grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high-grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real-time PCR analysis pointed to miR-375 as a biomarker for high-grade bladder cancer while miR-146a could identify low-grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy. (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 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 (d)(U)C Filtration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Biomarker Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells SW780 EV-harvesting Medium EV-depleted medium Preparation of EDS Not specified Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type JS-24.38 Pelleting: speed (g) 100000 Wash: volume per pellet (ml) 33 Wash: time (min) 60 Wash: Rotor Type JS-24.38 Wash: speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210101	1/6	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Welton, Joanne Louise	2016	29%
Study summary Full title Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array All authors Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton Journal J Extracell Vesicles Abstract Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide) EV-METRIC 29% (60th 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 Metastatic prostate cancer, failed treatment 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 Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-110 Pelleting: speed (g) 200000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Other;Spectrophotometry Detected EV-associated proteins SOMAscan multiplex assay Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210101	2/6	Homo sapiens	Blood plasma	(d)(U)C SEC (non-commercial) Filtration	Welton, Joanne Louise	2016	29%
Study summary Full title Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array All authors Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton Journal J Extracell Vesicles Abstract Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide) EV-METRIC 29% (60th 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 Mestatatic prostate cancer, prior treatment 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 Size-exclusion chromatography (non-commercial) Filtration Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Blood plasma Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-110 Pelleting: speed (g) 200000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 12 Sample volume/column (mL) 1.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Other;Spectrophotometry Detected EV-associated proteins SOMAscan multiplex assay Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210101	5/6	Homo sapiens	Urine	(d)(U)C Filtration SEC (non-commercial)	Welton, Joanne Louise	2016	29%
Study summary Full title Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array All authors Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton Journal J Extracell Vesicles Abstract Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide) EV-METRIC 29% (60th 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 Metastatic prostate cancer, failed treatment 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 Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 200000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 2.8 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Other;Spectrophotometry Detected EV-associated proteins SOMAscan multiplex assay Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210101	6/6	Homo sapiens	Urine	(d)(U)C Filtration SEC (non-commercial)	Welton, Joanne Louise	2016	29%
Study summary Full title Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array All authors Joanne Louise Welton, Paul Brennan, Mark Gurney, Jason Paul Webber, Lisa Kate Spary, David Gil Carton, Juan Manuel Falcón-Pérez, Sean Peter Walton, Malcolm David Mason, Zsuzsanna Tabi, Aled Clayton Journal J Extracell Vesicles Abstract Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasi (show more...)Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios. (hide) EV-METRIC 29% (60th 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 Mestatatic prostate cancer, prior treatment 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 Filtration Size-exclusion chromatography (non-commercial) Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods 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 Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: rotor type Type 70 Ti Pelleting: speed (g) 200000 Filtration steps 0.22µm or 0.2µm Size-exclusion chromatography Total column volume (mL) 2.8 Sample volume/column (mL) 0.5 Resin type Sepharose CL-2B Characterization: Protein analysis Protein Concentration Method Other;Spectrophotometry Detected EV-associated proteins SOMAscan multiplex assay Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210036	1/2	Homo sapiens	HT-29	(d)(U)C UF	Knol, Jaco C	2016	29%
Study summary Full title Peptide-mediated 'miniprep' isolation of extracellular vesicles is suitable for high-throughput proteomics All authors Jaco C Knol, Inge de Reus, Tim Schelfhorst, Robin Beekhof, Meike de Wit, Sander R Piersma, Thang V Pham, Egbert F Smit, Henk M W Verheul, Connie R Jiménez Journal EuPA Open Proteom. Abstract Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived (show more...)Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since EVs are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, they represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation are laborious, time-consuming, and require high inputs. Ghosh and co-workers recently described an isolation method utilizing Heat Shock Protein (HSP)-binding peptide Vn96 to aggregate HSP-decorated EVs, which can be performed at small 'miniprep' scale. Based on microscopic, immunoblot, and RNA sequencing analyses this method compared well with ultracentrifugation-mediated EV isolation, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cell-conditioned medium. Despite a 30-fold different scale (ultracentrifugation: 60 ml/Vn96-mediated aggregation: 2 ml) both methods yielded comparable numbers of identified proteins (3115/3085), with similar reproducibility of identification (72.5%/75.5%) and spectral count-based quantification (average CV: 31%/27%). EV fractions obtained with either method contained established EV markers and proteins linked to vesicle-related gene ontologies. Thus, Vn96 peptide-mediated aggregation is an advantageous, simple and rapid approach for EV isolation from small biological samples, enabling high-throughput analysis in a biomarker discovery setting. (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 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 Ultrafiltration Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HT-29 EV-harvesting Medium Serum free medium 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 SW 40 Ti Pelleting: speed (g) 114000 Wash: volume per pellet (ml) 13.2 Wash: time (min) 90 Wash: Rotor Type SW 40 Ti Wash: speed (g) 114000 Ultra filtration Cut-off size (kDa) 3 Membrane type Not specified Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210137	2/2	Homo sapiens	Serum	ExoQuick	Youn Lee, Joo	2016	28%
Study summary Full title Circulating exosomes from patients with systemic lupus erythematosus induce an proinflammatory immune response All authors Joo Youn Lee, Jin Kyun Park, Eun Young Lee, Eun Bong Lee, Yeong Wook Song Journal Arthritis Res Ther Abstract Background: Exosomes are involved in intercellular communication. The aim of this study was to inves (show more...)Background: Exosomes are involved in intercellular communication. The aim of this study was to investigate whether circulating exosomes effectively contribute to the inflammatory response in systemic lupus erythematosus (SLE). Methods: Exosomes were purified from SLE patients and healthy controls (HCs). Healthy peripheral blood mononuclear cells (PBMCs) were stimulated with exosomes isolated from SLE patients and HCs in the presence or absence of Toll-like receptor (TLR) inhibitors. Production of interferon (IFN)-α, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were measured. Correlation between exosome levels and SLE disease activity was examined. Results: The serum exosomes levels were significantly higher in SLE patients than in HCs. SLE exosomes induced a higher production of IFN-α, TNF-α, IL-1β, and IL-6 compared to healthy exosomes. SLE serum that was depleted of exosomes and SLE exosomes that were mechanically disrupted failed to induce any significant cytokine production. Exosome-mediated production of TNF-α, IL-1β, and IL-6 was decreased by the TLR4 antagonist, whereas that of IFN-α was suppressed by the TLR1/2, TLR7, and TLR9 antagonists. Exosome levels correlated with disease activity in SLE patients (rho = 0.846, p = 0.008). Conclusions: The circulating exosomes are immunologically active and their levels correlate with disease activity in SLE patients. The circulating exosomes might serve as novel biomarkers of SLE disease activity. (hide) EV-METRIC 28% (70th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. 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 Systemic lupus erythematosus 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 ExoQuick Protein markers EV: CD81/ CD63 non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Serum Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined ELISA Antibody details provided? No Detected EV-associated proteins CD63/ CD81 Characterization: Lipid analysis No EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 50-100

	EV220425	1/1	Homo sapiens	Blood plasma	(d)(U)C qEV	Vogel R	2016	25%
Study summary Full title A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing. All authors Vogel R, Coumans FA, Maltesen RG, Böing AN, Bonnington KE, Broekman ML, Broom MF, Buzás EI, Christiansen G, Hajji N, Kristensen SR, Kuehn MJ, Lund SM, Maas SL, Nieuwland R, Osteikoetxea X, Schnoor R, Scicluna BJ, Shambrook M, de Vrij J, Mann SI, Hill AF, Pedersen S Journal J Extracell Vesicles Abstract Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential dia (show more...)Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations. (hide) EV-METRIC 25% (55th 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 qEV Protein markers EV: CD9 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 Commercial kit qEV Other Name other separation method qEV Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 0-400 EV concentration Yes TRPS Report type Modus Reported size (nm) 70-130 EV concentration Yes EM EM-type Transmission-EM/ Immuno-EM EM protein CD9 Image type Close-up Report size (nm) 80-250

	EV220214	1/1	Homo sapiens	Adipose-derived mesenchymal stem cells	(d)(U)C Filtration UF ExoQuick	Hu L	2016	25%
Study summary Full title Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. All authors Hu L, Wang J, Zhou X, Xiong Z, Zhao J, Yu R, Huang F, Zhang H, Chen L Journal Sci Rep Abstract Prolonged healing and scar formation are two major challenges in the treatment of soft tissue trauma (show more...)Prolonged healing and scar formation are two major challenges in the treatment of soft tissue trauma. Adipose mesenchymal stem cells (ASCs) play an important role in tissue regeneration, and recent studies have suggested that exosomes secreted by stem cells may contribute to paracrine signaling. In this study, we investigated the roles of ASCs-derived exosomes (ASCs-Exos) in cutaneous wound healing. We found that ASCs-Exos could be taken up and internalized by fibroblasts to stimulate cell migration, proliferation and collagen synthesis in a dose-dependent manner, with increased genes expression of N-cadherin, cyclin-1, PCNA and collagen I, III. In vivo tracing experiments demonstrated that ASCs-Exos can be recruited to soft tissue wound area in a mouse skin incision model and significantly accelerated cutaneous wound healing. Histological analysis showed increased collagen I and III production by systemic administration of exosomes in the early stage of wound healing, while in the late stage, exosomes might inhibit collagen expression to reduce scar formation. Collectively, our findings indicate that ASCs-Exos can facilitate cutaneous wound healing via optimizing the characteristics of fibroblasts. Our results provide a new perspective and therapeutic strategy for the use of ASCs-Exos in soft tissue repair. (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 Filtration Ultrafiltration ExoQuick Protein markers EV: CD9/ CD63 non-EV: Tubulin/ Lamin A/C Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Adipose-derived mesenchymal stem cells EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.2 or 0.22 µm Ultra filtration Cut-off size (kDa) 100 Membrane type Regenerated cellulose Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Detected contaminants Tubulin/ Lamin A/C Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-100 EM EM-type Transmission-EM Image type Close-up, Wide-field

	EV220059	4/8	Homo sapiens	MCF-7	(d)(U)C Total Exosome Isolation	Song X	2016	25%
Study summary Full title Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases. All authors Song X, Ding Y, Liu G, Yang X, Zhao R, Zhang Y, Zhao X, Anderson GJ, Nie G Journal J Biol Chem Abstract Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocyte (show more...)Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. (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: TSG101/ HER-2/ EGFR/ GAPDH non-EV: None Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF-7 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Detected EV-associated proteins GAPDH/ EGFR/ HER-2/ TSG101 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV220054	5/14	Homo sapiens	MCF7	Total Exosome Isolation	Salony	2016	25%
Study summary Full title AKT Inhibition Promotes Nonautonomous Cancer Cell Survival. All authors Salony , Solé X, Alves CP, Dey-Guha I, Ritsma L, Boukhali M, Lee JH, Chowdhury J, Ross KN, Haas W, Vasudevan S, Ramaswamy S Journal Mol Cancer Ther Abstract Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being e (show more...)Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with subtherapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multiscale, molecular snapshot of this "AKT(low)" cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously upregulate a host of other proteins and metabolites posttranscriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication. (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 (shedding) microvesicle 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 Commercial method Protein markers EV: CD81/ TNFSF10/ CD63 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Separation Method Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TNFSF10/ CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220054	6/14	Homo sapiens	MCF7	Total Exosome Isolation	Salony	2016	25%
Study summary Full title AKT Inhibition Promotes Nonautonomous Cancer Cell Survival. All authors Salony , Solé X, Alves CP, Dey-Guha I, Ritsma L, Boukhali M, Lee JH, Chowdhury J, Ross KN, Haas W, Vasudevan S, Ramaswamy S Journal Mol Cancer Ther Abstract Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being e (show more...)Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with subtherapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multiscale, molecular snapshot of this "AKT(low)" cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously upregulate a host of other proteins and metabolites posttranscriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication. (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 Akti-1/2 Focus vesicles (shedding) microvesicle 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 Commercial method Protein markers EV: CD81/ TNFSF10/ CD63 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells MCF7 EV-harvesting Medium EV-depleted medium Separation Method Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TNFSF10/ CD63/ CD81 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220054	9/14	Homo sapiens	HCT116	Total Exosome Isolation	Salony	2016	25%
Study summary Full title AKT Inhibition Promotes Nonautonomous Cancer Cell Survival. All authors Salony , Solé X, Alves CP, Dey-Guha I, Ritsma L, Boukhali M, Lee JH, Chowdhury J, Ross KN, Haas W, Vasudevan S, Ramaswamy S Journal Mol Cancer Ther Abstract Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being e (show more...)Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with subtherapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multiscale, molecular snapshot of this "AKT(low)" cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously upregulate a host of other proteins and metabolites posttranscriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication. (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 (shedding) microvesicle 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 Commercial method Protein markers EV: CD81/ TNFSF10/ CD63 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116 EV-harvesting Medium EV-depleted medium Separation Method Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TNFSF10/ CD63/ CD81 Not detected EV-associated proteins CD81/ CD63 Not detected contaminants Calnexin/ GM130 Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220054	10/14	Homo sapiens	HCT116	Total Exosome Isolation	Salony	2016	25%
Study summary Full title AKT Inhibition Promotes Nonautonomous Cancer Cell Survival. All authors Salony , Solé X, Alves CP, Dey-Guha I, Ritsma L, Boukhali M, Lee JH, Chowdhury J, Ross KN, Haas W, Vasudevan S, Ramaswamy S Journal Mol Cancer Ther Abstract Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being e (show more...)Small molecule inhibitors of AKT (v-akt murine thymoma viral oncogene homolog) signaling are being evaluated in patients with various cancer types, but have so far proven therapeutically disappointing for reasons that remain unclear. Here, we treat cancer cells with subtherapeutic doses of Akti-1/2, an allosteric small molecule AKT inhibitor, in order to experimentally model pharmacologic inhibition of AKT signaling in vitro. We then apply a combined RNA, protein, and metabolite profiling approach to develop an integrated, multiscale, molecular snapshot of this "AKT(low)" cancer cell state. We find that AKT-inhibited cancer cells suppress thousands of mRNA transcripts, and proteins related to the cell cycle, ribosome, and protein translation. Surprisingly, however, these AKT-inhibited cells simultaneously upregulate a host of other proteins and metabolites posttranscriptionally, reflecting activation of their endo-vesiculo-membrane system, secretion of inflammatory proteins, and elaboration of extracellular microvesicles. Importantly, these microvesicles enable rapidly proliferating cancer cells of various types to better withstand different stress conditions, including serum deprivation, hypoxia, or cytotoxic chemotherapy in vitro and xenografting in vivo. These findings suggest a model whereby cancer cells experiencing a partial inhibition of AKT signaling may actually promote the survival of neighbors through non-cell autonomous communication. (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 Akti-1/2 Focus vesicles (shedding) microvesicle 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 Commercial method Protein markers EV: CD81/ TNFSF10/ CD63 non-EV: Calnexin/ GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HCT116 EV-harvesting Medium EV-depleted medium Separation Method Commercial kit Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins TNFSF10/ CD81/ CD63 Not detected contaminants GM130/ Calnexin Characterization: RNA analysis RNA analysis Type RNAsequencing Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210411	1/2	Rattus norvegicus	PC12	ExoQuick	Grindheim AK	2016	25%
Study summary Full title Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2. All authors Grindheim AK, Hollås H, Raddum AM, Saraste J, Vedeler A Journal J Cell Sci Abstract Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation a (show more...)Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation and functions are tightly regulated by its post-translational modifications. AnxA2 and its Tyr23-phosphorylated form (pTyr23AnxA2) are involved in malignant cell transformation, metastasis and angiogenesis. Here, we show that H2O2 exerts rapid, simultaneous and opposite effects on the Tyr23 phosphorylation status of AnxA2 in two distinct compartments of rat pheochromocytoma (PC12) cells. Reactive oxygen species induce dephosphorylation of pTyr23AnxA2 located in the PML bodies of the nucleus, whereas AnxA2 associated with F-actin at the cell cortex is Tyr23 phosphorylated. The H2O2-induced responses in both compartments are transient and the pTyr23AnxA2 accumulating at the cell cortex is subsequently incorporated into vesicles and then released to the extracellular space. Blocking nuclear export by leptomycin B does not affect the nuclear pool of pTyr23AnxA2, but increases the amount of total AnxA2 in this compartment, indicating that the protein might have several functions in the nucleus. These results suggest that Tyr23 phosphorylation can regulate the function of AnxA2 at distinct subcellular sites. (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 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 Commercial method Protein markers EV: TSG101/ CD63/ T-cadherin/ PTyr23AnxA2/ AnxA2 non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells PC12 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins TSG101 Not detected EV-associated proteins T-cadherin/ PTyr23AnxA2/ AnxA2/ CD63 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210411	2/2	Rattus norvegicus	PC12	ExoQuick	Grindheim AK	2016	25%
Study summary Full title Reactive oxygen species exert opposite effects on Tyr23 phosphorylation of the nuclear and cortical pools of annexin A2. All authors Grindheim AK, Hollås H, Raddum AM, Saraste J, Vedeler A Journal J Cell Sci Abstract Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation a (show more...)Annexin A2 (AnxA2) is a multi-functional and -compartmental protein whose subcellular localisation and functions are tightly regulated by its post-translational modifications. AnxA2 and its Tyr23-phosphorylated form (pTyr23AnxA2) are involved in malignant cell transformation, metastasis and angiogenesis. Here, we show that H2O2 exerts rapid, simultaneous and opposite effects on the Tyr23 phosphorylation status of AnxA2 in two distinct compartments of rat pheochromocytoma (PC12) cells. Reactive oxygen species induce dephosphorylation of pTyr23AnxA2 located in the PML bodies of the nucleus, whereas AnxA2 associated with F-actin at the cell cortex is Tyr23 phosphorylated. The H2O2-induced responses in both compartments are transient and the pTyr23AnxA2 accumulating at the cell cortex is subsequently incorporated into vesicles and then released to the extracellular space. Blocking nuclear export by leptomycin B does not affect the nuclear pool of pTyr23AnxA2, but increases the amount of total AnxA2 in this compartment, indicating that the protein might have several functions in the nucleus. These results suggest that Tyr23 phosphorylation can regulate the function of AnxA2 at distinct subcellular sites. (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 H2O2 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 Commercial method Protein markers EV: TSG101/ PTyr23AnxA2/ AnxA2/ T-cadherin/ CD63/ pTyr23AnxA2/ Ubiquitin non-EV: None Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells PC12 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Separation Method Commercial kit ExoQuick Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Detected EV-associated proteins CD63/ PTyr23AnxA2/ AnxA2/ TSG101 Not detected EV-associated proteins T-cadherin Detected EV-associated proteins pTyr23AnxA2/ Ubiquitin Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210318	3/12	Homo sapiens	Hs578T	(d)(U)C Total Exosome Isolation	Fiskaa T	2016	25%
Study summary Full title Distinct Small RNA Signatures in Extracellular Vesicles Derived from Breast Cancer Cell Lines. All authors Fiskaa T, Knutsen E, Nikolaisen MA, Jørgensen TE, Johansen SD, Perander M, Seternes OM Journal PLoS One Abstract Breast cancer is a heterogeneous disease, and different subtypes of breast cancer show distinct cell (show more...)Breast cancer is a heterogeneous disease, and different subtypes of breast cancer show distinct cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. Understanding the molecular features responsible for this heterogeneity is important for correct diagnosis and better treatment strategies. Extracellular vesicles (EVs) and their associated molecules have gained much attention as players in intercellular communication, ability to precondition specific organs for metastatic invasion, and for their potential role as circulating cancer biomarkers. EVs are released from the cells and contain proteins, DNA, and long and small RNA species. Here we show by high-throughput small RNA-sequencing that EVs from nine different breast cancer cell lines share common characteristics in terms of small RNA content that are distinct from their originating cells. Most strikingly, a highly abundant small RNA molecule derived from the nuclear 28S rRNA is vastly enriched in EVs. The miRNA profiles in EVs correlate with the cellular miRNA expression pattern, but with a few exceptions that includes miR-21. This cancer-associated miRNA is retained in breast cancer cell lines. Finally, we report that EVs from breast cancer cell lines cluster together based on their small RNA signature when compared to EVs derived from other cancer cell lines. Altogether, our data demonstrate that breast cancer cell lines manifest a specific small RNA signature in their released EVs. This opens up for further evaluation of EVs as breast cancer biomarkers. (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 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 Total Exosome Isolation Protein markers EV: CD63/ actin non-EV: None Proteomics no Show all info Study aim Biomarker/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Hs578T EV-harvesting Medium EV-depleted medium Preparation of EDS 12h at 120,000g Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Commercial kit Total Exosome Isolation Other Name other separation method Total Exosome Isolation Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63/ actin Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ RNA -sequencing Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Size range/distribution

	EV210219	4/4	Homo sapiens	Blood plasma	(d)(U)C	Moro, Laura	2016	25%
Study summary Full title Placental Microparticles and MicroRNAs in Pregnant Women with Plasmodium falciparum or HIV Infection All authors Laura Moro, Azucena Bardají, Eusebio Macete, Diana Barrios, Diana M Morales-Prieto, Carolina España, Inacio Mandomando, Betuel Sigaúque, Carlota Dobaño, Udo R Markert, Daniel Benitez-Ribas, Pedro L Alonso, Clara Menéndez, Alfredo Mayor Journal PLoS One Abstract Background: During pregnancy, syncytiotrophoblast vesicles contribute to maternal tolerance towards (show more...)Background: During pregnancy, syncytiotrophoblast vesicles contribute to maternal tolerance towards the fetus, but also to pathologies such as pre-eclampsia. The aim of the study was to address whether Plasmodium falciparum and HIV infections in pregnancy affect the secretion, microRNA content and function of trophoblast microparticles. Methods: Microparticles were isolated and characterized from 122 peripheral plasmas of Mozambican pregnant women, malaria- and/or HIV-infected and non-infected. Expression of placenta-related microRNAs in microparticles was analysed by qPCR and the effect of circulating microparticles on dendritic cells assessed by phenotype analysis and cytokine/chemokine measurement. Results: Concentrations of total and trophoblast microparticles detected by flow cytometry were higher in HIV-positive (P = 0.005 and P = 0.030, respectively) compared to non-infected mothers, as well as in women delivering low birthweight newborns (P = 0.032 and P = 0.021, respectively). miR-517c was overexpressed in mothers with placental malaria (P = 0.034), compared to non-infected. Microparticles from HIV-positive induced a higher expression of MHCII (P = 0.021) and lower production of MCP1 (P = 0.008) than microparticles from non-infected women. Conclusions: In summary, alterations in total and trophoblast microparticles associated with malaria and HIV in pregnant women may have an immunopathogenic role. The potential for placental-derived vesicles and microRNAs as biomarkers of adverse outcomes during pregnancy and malaria infection should be confirmed in future studies. (hide) EV-METRIC 25% (55th 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 Pregnant, positive for both placental malaria and HIV Focus vesicles microparticle 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: PSG1 non-EV: None Proteomics no Show all info Study aim Function/Identification of content (omics approaches) 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) 30 Pelleting: rotor type Not specified Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Not determined Flow cytometry Type of Flow cytometry BD LSRFortessa SORP Calibration bead size 0.2 and 1 Antibody details provided? No Detected EV-associated proteins PSG1 Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment No RNAse treatment No Characterization: Lipid analysis Yes, Characterization: Particle analysis EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 200-1000nm

	EV210099	3/9	Homo sapiens	HEK293 T	ExoQuick	Rider, Mark A	2016	25%
Study summary Full title ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of Extracellular Vesicles All authors Mark A Rider, Stephanie N Hurwitz, David G Meckes Jr Journal Sci Rep Abstract Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known (show more...)Initially thought to be a means for cells to eliminate waste, secreted extracellular vesicles, known as exosomes, are now understood to mediate numerous healthy and pathological processes. Though abundant in biological fluids, purifying exosomes has been challenging because their biophysical properties overlap with other secreted cell products. Easy-to-use commercial kits for harvesting exosomes are now widely used, but the relative low-purity and high-cost of the preparations restricts their utility. Here we describe a method for purifying exosomes and other extracellular vesicles by adapting methods for isolating viruses using polyethylene glycol. This technique, called ExtraPEG, enriches exosomes from large volumes of media rapidly and inexpensively using low-speed centrifugation, followed by a single small-volume ultracentrifugation purification step. Total protein and RNA harvested from vesicles is sufficient in quantity and quality for proteomics and sequencing analyses, demonstrating the utility of this method for biomarker discovery and diagnostics. Additionally, confocal microscopy studies suggest that the biological activity of vesicles is not impaired. The ExtraPEG method can be easily adapted to enrich for different vesicle populations, or as an efficient precursor to subsequent purification techniques, providing a means to harvest exosomes from many different biological fluids and for a wide variety of purposes. (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 ExoQuick Protein markers EV: CD63/ TSG101/ HSP70/ Alix non-EV: None Proteomics no Show all info Study aim New methodological development/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK293 T EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Separation Method Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Fluorometric assay (e.g. Qubit, NanoOrange,...) Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ TSG101/ HSP70/ Alix Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Not Reported Particle yield NA NA

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