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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120099	2/2	Mus musculus	NAY	(d)(U)C DG	Yuyama K	2012	44%
Study summary Full title Sphingolipid-modulated exosome secretion promotes clearance of amyloid-beta by microglia All authors Yuyama K, Sun H, Mitsutake S, Igarashi Y Journal J Biol Chem Abstract Amyloid ?-peptide (A?), the pathogenic agent of Alzheimer disease, is a physiological metabolite who (show more...)Amyloid ?-peptide (A?), the pathogenic agent of Alzheimer disease, is a physiological metabolite whose levels are constantly controlled in normal brain. Recent studies have demonstrated that a fraction of extracellular A? is associated with exosomes, small membrane vesicles of endosomal origin, although the fate of A? in association with exosome is largely unknown. In this study, we identified novel roles for neuron-derived exosomes acting on extracellular A?, i.e. exosomes drive conformational changes in A? to form nontoxic amyloid fibrils and promote uptake of A? by microglia. The A? internalized together with exosomes was further transported to lysosomes and degraded. We also found that blockade of phosphatidylserine on the surface of exosomes by annexin V not only prevented exosome uptake but also suppressed A? incorporation into microglia. In addition, we demonstrated that secretion of neuron-derived exosomes was modulated by the activities of sphingolipid-metabolizing enzymes, including neutral sphingomyelinase 2 (nSMase2) and sphingomyelin synthase 2 (SMS2). In transwell experiments, up-regulation of exosome secretion from neuronal cells by treatment with SMS2 siRNA enhanced A? uptake into microglial cells and significantly decreased extracellular levels of A?. Our findings indicate a novel mechanism responsible for clearance of A? through its association with exosomes. The modulation of the vesicle release and/or elimination may alter the risk of AD. (hide) EV-METRIC 44% (84th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C DG Protein markers EV: Alix/ TSG101/ GM1 non-EV: Cell organelle protein Proteomics no EV density (g/ml) 1.12-1.16 Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Density gradient Only used for validation of main results Yes Lowest density fraction 0.25 Highest density fraction 2.2999999999999998 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ GM1 Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins GM1

	EV120099	1/2	Mus musculus	NAY	(d)(U)C	Yuyama K	2012	11%
Study summary Full title Sphingolipid-modulated exosome secretion promotes clearance of amyloid-beta by microglia All authors Yuyama K, Sun H, Mitsutake S, Igarashi Y Journal J Biol Chem Abstract Amyloid ?-peptide (A?), the pathogenic agent of Alzheimer disease, is a physiological metabolite who (show more...)Amyloid ?-peptide (A?), the pathogenic agent of Alzheimer disease, is a physiological metabolite whose levels are constantly controlled in normal brain. Recent studies have demonstrated that a fraction of extracellular A? is associated with exosomes, small membrane vesicles of endosomal origin, although the fate of A? in association with exosome is largely unknown. In this study, we identified novel roles for neuron-derived exosomes acting on extracellular A?, i.e. exosomes drive conformational changes in A? to form nontoxic amyloid fibrils and promote uptake of A? by microglia. The A? internalized together with exosomes was further transported to lysosomes and degraded. We also found that blockade of phosphatidylserine on the surface of exosomes by annexin V not only prevented exosome uptake but also suppressed A? incorporation into microglia. In addition, we demonstrated that secretion of neuron-derived exosomes was modulated by the activities of sphingolipid-metabolizing enzymes, including neutral sphingomyelinase 2 (nSMase2) and sphingomyelin synthase 2 (SMS2). In transwell experiments, up-regulation of exosome secretion from neuronal cells by treatment with SMS2 siRNA enhanced A? uptake into microglial cells and significantly decreased extracellular levels of A?. Our findings indicate a novel mechanism responsible for clearance of A? through its association with exosomes. The modulation of the vesicle release and/or elimination may alter the risk of AD. (hide) EV-METRIC 11% (30th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin NAY Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Protein markers EV: Alix/ TSG101/ GM1 non-EV: Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ TSG101/ GM1 ELISA Antibody details provided? No Detected EV-associated proteins GM1 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV120099
species	Mus musculus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG	(d)(U)C
Exp. nr.	2	1
EV-METRIC %	44	11