Glycosylation, a vital protein modification that plays a important part in ligand-binding recognition, could influence the affinity of EVs for diverse tissues. Approaches: Purified EVs derived from hepatic cells have been treated with a neuraminidase, an enzyme that digests the terminal sialic acid residues from glycoproteins. Afterwards, EVs had been labelled with [124I]NaI and injected in mice intravenously or inside the hook (the lateral tarsal area just above the ankle). The volume of radioactivity in significant organs was measured at Serine/Threonine-Protein Kinase 26 Proteins Purity & Documentation different time points immediately after administration each in vivo making use of positron emission tomography and ex vivo (immediately after animal sacrifice) applying dissection and gamma counting. Results: As anticipated, intravenous injection leads to fast accumulation of EVs within the liver, Cathepsin L1 Proteins Biological Activity contrary to [124I]NaI (no EVs, utilised because the control). Following some hours, the distribution leads to the presence of EVs in distinctive organs, and interestingly, also in brain. Glycosidase-treated EVs showed an important accumulation in the lungs compared with intact EVs. This pattern was also confirmed within the animals injected by means of the hook.ISEV 2018 abstract bookSummary/Conclusion: The EVs derived from hepatic cell lines are systemically distributed in many organs, though the principle accumulation occurs in the liver. The modification with the glycome that decorates the EVs surface affects the distribution of these vesicles, enabling the transformed EVs to reach far more abundantly the lungs. Additional research will aid to establish different protocols to target a variety of organs. Funding: This perform was supported by RAMON ARECES FUNDATION plus the Spanish Ministry of Economy and Competitiveness MINECO (Plan NACIONAL).PS03.A quantitative strategy to measure EV uptake Victor Toribio1; Beatriz Carde s2; Sara Morales-Lopez3; Soraya L ezMart four; Carlos Caba s2; Mar Y ez-M Centro de Biolog Molecular “Severo Ochoa” CSIC/UAM, Madrid, Spain; CBM-SO, CSIC, Madrid, Spain; 3Instituto de Investigaci Sanitaria Princesa (IIS-IP), Madrid, Spain; 4Molecular Biology Center Severo Ochoa (CBM), Instituto de Investigaci Sanitaria Princesa (IIS-IP), Madrid, Spain; five Departamento de Biolog Molecular, UAM, Madrid, Spain1Background: Because EV size lies beneath the limit of resolution of optical procedures, discrimination among EV binding towards the target cell and uptake is generally not feasible with microscopy or cytometry procedures, top to artefactual benefits. Our aim was to construct a appropriate and quantitative process to analyse and discover the molecular mechanisms of EV uptake by the target cells, determined by tetraspanins, classical EV-markers. Methods: Human tetraspanins CD9 and CD63 had been fused to a dual GFP-Luciferase-split vector tag. Incorporation of fusion proteins into EVs was assessed by bead-based flow cytometry and Western blot. Measurement of binding and uptake was performed by a mixture of classical Renilla substrates and Enduren. Benefits: Dual GFP-Luciferase-split constructs of tetraspanins have been shown to present the same subcellular localization than endogenous proteins. Furthermore, by both bead-based flow cytometry and Western blot they could be correctly detected at EVs immediately after lentiviral infection of making cells. Incubation of target cells that expressed the complementary domains of the dual GFP-Luciferase-split construct with transfected exosomes could not recover the fluorescence or the luciferase function. However, when EVs carried the completely reconstituted DualGFP-Lucife.