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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120122	1/1	Homo sapiens Mus musculus	NAY	(d)(U)C Filtration	Danzer KM	2012	11%
Study summary Full title Exosomal cell-to-cell transmission of alpha synuclein oligomers All authors Danzer KM, Kranich LR, Ruf WP, Cagsal-Getkin O, Winslow AR, Zhu L, Vanderburg CR, McLean PJ Journal Mol Neurodegener Abstract BACKGROUND: Aggregation of alpha-synuclein (?syn) and resulting cytotoxicity is a hallmark of sporad (show more...)BACKGROUND: Aggregation of alpha-synuclein (?syn) and resulting cytotoxicity is a hallmark of sporadic and familial Parkinson's disease (PD) as well as dementia with Lewy bodies, with recent evidence implicating oligomeric and pre-fibrillar forms of ?syn as the pathogenic species. Recent in vitro studies support the idea of transcellular spread of extracellular, secreted ?syn across membranes. The aim of this study is to characterize the transcellular spread of ?syn oligomers and determine their extracellular location. RESULTS: Using a novel protein fragment complementation assay where ?syn is fused to non-bioluminescent amino-or carboxy-terminus fragments of humanized Gaussia Luciferase we demonstrate here that ?syn oligomers can be found in at least two extracellular fractions: either associated with exosomes or free. Exosome-associated ?syn oligomers are more likely to be taken up by recipient cells and can induce more toxicity compared to free ?syn oligomers. Specifically, we determine that ?syn oligomers are present on both the outside as well as inside of exosomes. Notably, the pathway of secretion of ?syn oligomers is strongly influenced by autophagic activity. CONCLUSIONS: Our data suggest that ?syn may be secreted via different secretory pathways. We hypothesize that exosome-mediated release of ?syn oligomers is a mechanism whereby cells clear toxic ?syn oligomers when autophagic mechanisms fail to be sufficient. Preventing the early events in ?syn exosomal release and uptake by inducing autophagy may be a novel approach to halt disease spreading in PD and other synucleinopathies. (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 Filtration Protein markers EV: Alix/ Flotillin non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens / Mus musculus Sample Type Cell culture supernatant EV-harvesting Medium EV Depleted Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins Alix/ Flotillin ELISA Antibody details provided? No Detected EV-associated proteins Flotillin Characterization: Particle analysis EM EM-type transmission EM/ immune EM EM protein CD63 Image type Wide-field

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EV-TRACK ID	EV120122
species	Homo sapiens Mus musculus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	11