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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV110027	2/2	Rattus norvegicus/rattus	Cell culture supernatant	DG (d)(U)C Filtration	Lachenal G	2011	44%
Study summary Full title Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity All authors Lachenal G, Pernet-Gallay K, Chivet M, Hemming FJ, Belly A, Bodon G, Blot B, Haase G, Goldberg Y, Sadoul R Journal Mol Cell Neurosci Abstract Exosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane (show more...)Exosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane of endosomal intermediates called multivesicular bodies. They represent ways for discarding proteins and metabolites and also for intercellular transfer of proteins and RNAs. In the nervous system, it has been hypothesized that exosomes might be involved in the normal physiology of the synapse and possibly allow the trans-synaptic propagation of pathogenic proteins throughout the tissue. As a first step to validate this concept, we used biochemical and morphological approaches to demonstrate that mature cortical neurons in culture do indeed secrete exosomes. Using electron microscopy, we observed exosomes being released from somato-dendritic compartments. The endosomal origin of exosomes was demonstrated by showing that the C-terminal domain of tetanus toxin specifically endocytosed by neurons and accumulating inside multivesicular bodies, is released in the extracellular medium in association with exosomes. Finally, we found that exosomal release is modulated by glutamatergic synaptic activity, suggesting that this process might be part of normal synaptic physiology. Thus, our study paves the way towards the demonstration that exosomes take part in the physiology of the normal and pathological nervous system. (hide) EV-METRIC 44% (78th 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 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 DNF Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography DG + (d)(U)C + Filtration Protein markers EV: Alix/ GluR2/3/ Flotilin1/ L1CAM non-EV: Proteomics no EV density (g/ml) 1.09-1.15 Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 50 Density gradient Only used for validation of main results 1 Density medium Sucrose Lowest density fraction 0.21099999999999999 Highest density fraction 2.2549999999999999 Speed (g) 100000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ Flotilin1/ L1CAM/ GluR2/3 ELISA Detected EV-associated proteins L1CAM/ GluR2/3 Characterization: Particle analysis EM EM-type immune EM Proteïns L1CAM;GluR2 Image type Close-up, Wide-field

	EV110027	1/2	Rattus norvegicus/rattus	Cell culture supernatant	(d)(U)C Filtration	Lachenal G	2011	0%
Study summary Full title Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity All authors Lachenal G, Pernet-Gallay K, Chivet M, Hemming FJ, Belly A, Bodon G, Blot B, Haase G, Goldberg Y, Sadoul R Journal Mol Cell Neurosci Abstract Exosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane (show more...)Exosomes are microvesicles released into the extracellular medium upon fusion to the plasma membrane of endosomal intermediates called multivesicular bodies. They represent ways for discarding proteins and metabolites and also for intercellular transfer of proteins and RNAs. In the nervous system, it has been hypothesized that exosomes might be involved in the normal physiology of the synapse and possibly allow the trans-synaptic propagation of pathogenic proteins throughout the tissue. As a first step to validate this concept, we used biochemical and morphological approaches to demonstrate that mature cortical neurons in culture do indeed secrete exosomes. Using electron microscopy, we observed exosomes being released from somato-dendritic compartments. The endosomal origin of exosomes was demonstrated by showing that the C-terminal domain of tetanus toxin specifically endocytosed by neurons and accumulating inside multivesicular bodies, is released in the extracellular medium in association with exosomes. Finally, we found that exosomal release is modulated by glutamatergic synaptic activity, suggesting that this process might be part of normal synaptic physiology. Thus, our study paves the way towards the demonstration that exosomes take part in the physiology of the normal and pathological nervous system. (hide) EV-METRIC 0% (median: 22% 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 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 DNF Focus vesicles exosomes Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography (d)(U)C + Filtration Protein markers EV: Alix/ Flotilin1 non-EV: Proteomics no Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method Differential ultracentrifugation 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 Obtain an EV pellet : Yes Pelleting: time(min) 50 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Western Blot Detected EV-associated proteins Alix/ Flotilin1 Characterization: Particle analysis

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