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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210312	1/4	Rattus norvegicus	Primary microglia	(d)(U)C	Gabrielli M	2015	22%
Study summary Full title Active endocannabinoids are secreted on extracellular membrane vesicles. All authors Gabrielli M, Battista N, Riganti L, Prada I, Antonucci F, Cantone L, Matteoli M, Maccarrone M, Verderio C Journal EMBO Rep Abstract Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To e (show more...)Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids. (hide) EV-METRIC 22% (59th 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 ATP 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: Flotillin1/ Alix/ TSG101 non-EV: SP1/ TOM20/ GS28 Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells Primary microglia EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: speed (g) 10000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ TSG101 Not detected EV-associated proteins Adipophilin Detected contaminants SP1/ TOM20/ GS28 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 334.4 EV concentration Yes Particle yield as number of particles per million cells: 7.39e+8

	EV210312	2/4	Rattus norvegicus	Primary microglia	(d)(U)C	Gabrielli M	2015	22%
Study summary Full title Active endocannabinoids are secreted on extracellular membrane vesicles. All authors Gabrielli M, Battista N, Riganti L, Prada I, Antonucci F, Cantone L, Matteoli M, Maccarrone M, Verderio C Journal EMBO Rep Abstract Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To e (show more...)Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids. (hide) EV-METRIC 22% (59th 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 ATP 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: Flotillin1/ Alix/ TSG101 non-EV: SP1/ TOM20/ GS28 Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus Sample Type Cell culture supernatant EV-producing cells Primary microglia EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Antibody details provided? No Antibody dilution provided? Yes Detected EV-associated proteins Flotillin1/ Alix/ TSG101 Not detected EV-associated proteins Adipophilin Detected contaminants SP1/ TOM20/ GS28 Characterization: Lipid analysis Yes Characterization: Particle analysis NTA Report type Mean Reported size (nm) 168.7 EV concentration Yes Particle yield as number of particles per million cells: 7.66e+11

	EV210312	3/4	Mus musculus	N9	(d)(U)C	Gabrielli M	2015	0%
Study summary Full title Active endocannabinoids are secreted on extracellular membrane vesicles. All authors Gabrielli M, Battista N, Riganti L, Prada I, Antonucci F, Cantone L, Matteoli M, Maccarrone M, Verderio C Journal EMBO Rep Abstract Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To e (show more...)Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids. (hide) EV-METRIC 0% (median: 14% of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis 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 ATP 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 no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells N9 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Pelleting performed Yes Pelleting: speed (g) 10000 Characterization: Protein analysis None Protein Concentration Method microBCA Characterization: Lipid analysis Yes Characterization: Particle analysis None

	EV210312	4/4	Mus musculus	N9	(d)(U)C	Gabrielli M	2015	0%
Study summary Full title Active endocannabinoids are secreted on extracellular membrane vesicles. All authors Gabrielli M, Battista N, Riganti L, Prada I, Antonucci F, Cantone L, Matteoli M, Maccarrone M, Verderio C Journal EMBO Rep Abstract Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To e (show more...)Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB1), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids. (hide) EV-METRIC 0% (median: 14% of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis 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 ATP 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 no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells N9 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: speed (g) 100000 Characterization: Protein analysis None Protein Concentration Method microBCA Characterization: Lipid analysis Yes Characterization: Particle analysis None

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EV-TRACK ID	EV210312
species	Rattus norvegicus	Rattus norvegicus	Mus musculus	Mus musculus
sample type	Cell culture	Cell culture	Cell culture	Cell culture
cell type	Primary microglia	Primary microglia	N9	N9
condition	ATP	ATP	ATP	ATP
separation protocol	dUC	dUC	dUC	dUC
vesicle related term	(shedding) microvesicle	exosome	exosome	(shedding) microvesicle
Exp. nr.	1	2	3	4
EV-METRIC %	22	22	0	0