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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV120040	1/1	Rattus norvegicus/rattus	NAY	(d)(U)C DC	Rana S	2012	22%
Study summary Full title Toward tailored exosomes: the exosomal tetraspanin web contributes to target cell selection All authors Rana S, Yue S, Stadel D, Zöller M Journal Int J Biochem Cell Biol Abstract Exosomes are discussed as potent therapeutics due to efficient transfer of proteins, mRNA and miRNA (show more...)Exosomes are discussed as potent therapeutics due to efficient transfer of proteins, mRNA and miRNA in selective targets. However, therapeutic exosome application requires knowledge on target structures to avoid undue delivery. Previous work suggesting exosomal tetraspanin-integrin complexes to be involved in target cell binding, we aimed to control this hypothesis and to define target cell ligands. Exosomes are rich in tetraspanins that associate besides other molecules with integrins. Co-immunoprecipitation of exosome lysates from rat tumor lines that differ only with respect to Tspan8 and beta4 revealed promiscuity of tetraspanin-integrin associations, but also few preferential interactions like that of Tspan8 with alpha4 and beta4 integrin chains. These minor differences in exosomal tetraspanin-complexes strongly influence target cell selection in vitro and in vivo, efficient exosome-uptake being seen in hematopoietic cells and solid organs. Exosomes expressing the Tspan8-alpha4 complex are most readily taken up by endothelial and pancreas cells, CD54 serving as a major ligand. Selectivity of uptake was confirmed with exosomes from an alpha4 cDNA transfected Tspan8(+) lymph node stroma line. Distinct from exosomes from the parental line, the latter preferentially targeted endothelial cells and in vivo the pancreas. Importantly, pulldown experiments provided strong evidence that exosome-uptake occurs in internalization-prone membrane domains. This is the first report on the exosomal tetraspanin web contributing to target cell selection such that predictions can be made on potential targets, which will facilitate tailoring exosomes for drug delivery. (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 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 DC Protein markers EV: Clathrin/ Thrombospondin/ Clusterin/ Rab7/ Tubulin/ Tspan/ Neuropilin/ Annexin2/ HSP70/ GDF15/ CD9 non-EV: Cell organelle protein Proteomics yes Show all info Study aim Function Sample Species Rattus norvegicus/rattus Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g 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) 90 Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ HSP70/ Clusterin/ GDF15/ Neuropilin/ Thrombospondin/ Rab7/ Tspan/ Annexin2/ Tubulin/ Clathrin Detected contaminants Cell organelle protein ELISA Antibody details provided? No Detected EV-associated proteins Clusterin/ GDF15/ Neuropilin/ Thrombospondin/ Rab7/ Tspan/ Annexin2/ Tubulin/ Clathrin Flow cytometry specific beads Antibody details provided? No Antibody dilution provided? No Selected surface protein(s) Yes

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EV-TRACK ID	EV120040
species	Rattus norvegicus/rattus
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C DC
Exp. nr.	1
EV-METRIC %	22