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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210493	1/4	Homo sapiens	hCMEC/D3	(d)(U)C	Dozio V	2017	33%
Study summary Full title Characterisation of extracellular vesicle-subsets derived from brain endothelial cells and analysis of their protein cargo modulation after TNF exposure. All authors Dozio V, Sanchez JC Journal J Extracell Vesicles Abstract Little is known about the composition and functional differences between extracellular vesicle (EV) (show more...)Little is known about the composition and functional differences between extracellular vesicle (EV) subsets, such as microvesicles (MVs) and exosomes (EXOs), nor to what extent their cargo reflects the phenotypic state of the cell of origin. Brain endothelial cells are the constitutive part of the blood-brain barrier (BBB), a selective barrier that maintains brain homeostasis. BBB impairment is associated with several neuroinflammatory diseases with the pro-inflammatory cytokine tumour necrosis factor (TNF) often playing a key role. In the present study, shotgun proteomics and parallel reaction monitoring (PRM)-based targeted mass spectrometry were used to characterise brain endothelial cell-released EVs, and to study how TNF exposure modulated EV protein cargoes. MVs were found to be enriched in mitochondrial and cytoskeletal proteins, whereas EXOs were enriched in adhesion, histone and ribosomal proteins. After stimulation with TNF, several proteins involved in TNF and NF-κB signalling pathways, that were found to be differentially expressed in cells, were also differentially expressed in both MVs and EXOs. Thus, our results revealed some novel proteins as potentially useful candidates for discriminating between MVs and EXOs, together with additional evidence that cells "package" proteins in EVs systematically and according to their phenotypic state. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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 (Differential) (ultra)centrifugation Protein markers EV: Alix non-EV: GRP94 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hCMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 18000 Wash: time (min) 30 Wash: Rotor Type SW 55 Ti Wash: speed (g) 18000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins Alix Detected contaminants GRP94 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 215/ 375 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 150-300

	EV210493	2/4	Homo sapiens	hCMEC/D3	(d)(U)C	Dozio V	2017	33%
Study summary Full title Characterisation of extracellular vesicle-subsets derived from brain endothelial cells and analysis of their protein cargo modulation after TNF exposure. All authors Dozio V, Sanchez JC Journal J Extracell Vesicles Abstract Little is known about the composition and functional differences between extracellular vesicle (EV) (show more...)Little is known about the composition and functional differences between extracellular vesicle (EV) subsets, such as microvesicles (MVs) and exosomes (EXOs), nor to what extent their cargo reflects the phenotypic state of the cell of origin. Brain endothelial cells are the constitutive part of the blood-brain barrier (BBB), a selective barrier that maintains brain homeostasis. BBB impairment is associated with several neuroinflammatory diseases with the pro-inflammatory cytokine tumour necrosis factor (TNF) often playing a key role. In the present study, shotgun proteomics and parallel reaction monitoring (PRM)-based targeted mass spectrometry were used to characterise brain endothelial cell-released EVs, and to study how TNF exposure modulated EV protein cargoes. MVs were found to be enriched in mitochondrial and cytoskeletal proteins, whereas EXOs were enriched in adhesion, histone and ribosomal proteins. After stimulation with TNF, several proteins involved in TNF and NF-κB signalling pathways, that were found to be differentially expressed in cells, were also differentially expressed in both MVs and EXOs. Thus, our results revealed some novel proteins as potentially useful candidates for discriminating between MVs and EXOs, together with additional evidence that cells "package" proteins in EVs systematically and according to their phenotypic state. (hide) EV-METRIC 33% (75th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin 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: Alix non-EV: GRP94 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hCMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins Alix Not detected contaminants GRP94 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 150 EM EM-type Transmission-EM Image type Wide-field Report size (nm) 40-120

	EV210493	3/4	Homo sapiens	hCMEC/D3	(d)(U)C	Dozio V	2017	29%
Study summary Full title Characterisation of extracellular vesicle-subsets derived from brain endothelial cells and analysis of their protein cargo modulation after TNF exposure. All authors Dozio V, Sanchez JC Journal J Extracell Vesicles Abstract Little is known about the composition and functional differences between extracellular vesicle (EV) (show more...)Little is known about the composition and functional differences between extracellular vesicle (EV) subsets, such as microvesicles (MVs) and exosomes (EXOs), nor to what extent their cargo reflects the phenotypic state of the cell of origin. Brain endothelial cells are the constitutive part of the blood-brain barrier (BBB), a selective barrier that maintains brain homeostasis. BBB impairment is associated with several neuroinflammatory diseases with the pro-inflammatory cytokine tumour necrosis factor (TNF) often playing a key role. In the present study, shotgun proteomics and parallel reaction monitoring (PRM)-based targeted mass spectrometry were used to characterise brain endothelial cell-released EVs, and to study how TNF exposure modulated EV protein cargoes. MVs were found to be enriched in mitochondrial and cytoskeletal proteins, whereas EXOs were enriched in adhesion, histone and ribosomal proteins. After stimulation with TNF, several proteins involved in TNF and NF-κB signalling pathways, that were found to be differentially expressed in cells, were also differentially expressed in both MVs and EXOs. Thus, our results revealed some novel proteins as potentially useful candidates for discriminating between MVs and EXOs, together with additional evidence that cells "package" proteins in EVs systematically and according to their phenotypic state. (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 TNF 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 (Differential) (ultra)centrifugation Protein markers EV: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hCMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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 Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 18000 Wash: time (min) 30 Wash: Rotor Type SW 55 Ti Wash: speed (g) 18000 Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

	EV210493	4/4	Homo sapiens	hCMEC/D3	(d)(U)C	Dozio V	2017	29%
Study summary Full title Characterisation of extracellular vesicle-subsets derived from brain endothelial cells and analysis of their protein cargo modulation after TNF exposure. All authors Dozio V, Sanchez JC Journal J Extracell Vesicles Abstract Little is known about the composition and functional differences between extracellular vesicle (EV) (show more...)Little is known about the composition and functional differences between extracellular vesicle (EV) subsets, such as microvesicles (MVs) and exosomes (EXOs), nor to what extent their cargo reflects the phenotypic state of the cell of origin. Brain endothelial cells are the constitutive part of the blood-brain barrier (BBB), a selective barrier that maintains brain homeostasis. BBB impairment is associated with several neuroinflammatory diseases with the pro-inflammatory cytokine tumour necrosis factor (TNF) often playing a key role. In the present study, shotgun proteomics and parallel reaction monitoring (PRM)-based targeted mass spectrometry were used to characterise brain endothelial cell-released EVs, and to study how TNF exposure modulated EV protein cargoes. MVs were found to be enriched in mitochondrial and cytoskeletal proteins, whereas EXOs were enriched in adhesion, histone and ribosomal proteins. After stimulation with TNF, several proteins involved in TNF and NF-κB signalling pathways, that were found to be differentially expressed in cells, were also differentially expressed in both MVs and EXOs. Thus, our results revealed some novel proteins as potentially useful candidates for discriminating between MVs and EXOs, together with additional evidence that cells "package" proteins in EVs systematically and according to their phenotypic state. (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 TNF 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: None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells hCMEC/D3 EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS 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) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method Bradford Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV210493
species	Homo sapiens
sample type	Cell culture
cell type	hCMEC/D3
condition	Control condition	Control condition	TNF	TNF
separation protocol	dUC	dUC	dUC	dUC
vesicle related term	(shedding) microvesicle	exosome	(shedding) microvesicle	exosome
Exp. nr.	1	2	3	4
EV-METRIC %	33	33	29	29