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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV100042	1/1	Canis familiaris	NAY	(d)(U)C DG	Komatsu T	2010	33%
Study summary Full title Extrusion of Na,K-ATPase and transferrin receptor with lipid raft-associated proteins in different populations of exosomes during reticulocyte maturation in dogs All authors Komatsu T, Arashiki N, Otsuka Y, Sato K, Inaba M Journal Jpn J Vet Res Abstract The present study characterizes canine reticulocyte exosomes. Exosomes are small membrane vesicles i (show more...)The present study characterizes canine reticulocyte exosomes. Exosomes are small membrane vesicles involved in membrane remodeling that are released from reticulocytes during the final maturation step of red blood cells. The vesicles collected from reticulocyte culture supernatants by differential centrifugation contained major exosomal proteins including heat shock protein cognate 70 (Hsc70) and transferrin receptors (TfR), consistent with the definition of the exosome. In addition, the Na,K-ATPase alpha-subunit and stomatin, a lipid raft-associated protein, were extruded by the exosome pathway, possibly leading to the absence of these proteins in erythrocytes, while the major protein constituents of erythrocyte membranes, spectrin and band 3 were retained in reticulocytes and not expelled into exosomes. The Na,K-ATPase alpha-subunit, as well as TfR and about half of the stomatin contained in exosomes, was obtained in a detergent-soluble fraction that was distinct from the lipid raft microdomain. Moreover, Na,K-ATPase and a portion of stomatin were distributed differently to Hsc70, TfR, stomatin, and ganglioside GM1 in vesicles separated with sucrose density gradient centrifugation. These results demonstrate that a heterogeneous group of exosomes participates in the loss of Na,K-ATPase and membrane remodeling during reticulocyte maturation in dogs. (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 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: HSC70/ Flotillin2 non-EV: Proteomics yes EV density (g/ml) 1.06-1.14 Show all info Study aim Biogenesis/Sorting Sample Species Canis familiaris Sample Type Cell culture supernatant Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps 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 10 Highest density fraction 35 Orientation Top-down Speed (g) 100000 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins HSC70/ Flotillin2 ELISA Antibody details provided? No Detected EV-associated proteins HSC70/ Flotillin2

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EV-TRACK ID	EV100042
species	Canis familiaris
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DG
Exp. nr.	1
EV-METRIC %	33