o:37143 Monitoring extracellular vesicle surface glyco-properties using fluorescent lectins and nanoparticle tracking analysis en Extracellular vesicles (EVs) are small particles released by all cell types. Different EVs isolation methods are widely used, yet none achieve an optimal balance between yield, purity, and structural integrity. This study aimed to establish a comparative approach for evaluating different EVs preparations using nanoparticle tracking analysis (NTA). Simple one-step assay relying on fluorescence-based NTA was used to evaluate lectin-binding to EVs as a measure of possible changes in their surface glycosylation during various isolation methods. Seminal EVs (sEVs) were isolated from normozoospermic men using ultracentrifugation alone - UC-sEVs or combined with size exclusion chromatography - UC-SEC-sEVs or microfiltration - UC-MF-sEVs. They were analyzed based on their size and lectin-binding properties using wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA I). While total sEVs and tetraspanin-positive sEVs maintained similar size distributions across all isolates, lectin-positive sEVs displayed a shift toward larger than 200 nm sEVs in UC-SEC-sEVs and UC-MF-sEVs, as compared to UC-sEVs. The ratio of larger (>200 nm) to smaller (30-200 nm) lectin-positive sEVs was increased, particularly for WGA in UC-MF-sEVs and RCA I in UC-SEC-sEVs. These findings demonstrate that SEC and MF combined with UC influence sEVs surface glycosylation and alters lectin-binding across EVs of different sizes. extracellular vesicles, fluorescence nanoparticle tracking analysis, ultracentrifugation, size exclusion chromatography, filtration, lectins 1552099 https://doi.org/10.1098/rsob.250136 2025-11-27T12:10:15.718Z 44 yes 46 Ninoslav Mitić University of Belgrade, Institute for the Application of Nuclear Energy, Serbia doktor bioloških nauka 0000-0003-2756-6517 Filip Janjić University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia doktor medicinskih nauka – veterinarska medicina person 0000-0002-5121-1145 Jelena Danilović-Luković Department of Life Sciences, University of Belgrade – Institute for Multidisciplinary Research, Belgrade, Serbia person Sanja Goč University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia doktor bioloških nauka person 0000-0002-6104-693X Tamara Janković University of Belgrade, Institute for the Application of Nuclear Energy INEP, Serbia Magistar farmacije-medicinski biohemičar person 0000-0002-8188-0921 Miroslava Janković Institute for the Application of Nuclear Energy INEP, University of Belgrade, Serbia doktor bioloških nauka person 0000-0002-7889-5110 application/pdf 1439168 https://phaidrabg.bg.ac.rs/o:37143 no yes 16 70 92000001 11A29 11A2903 11A27 11A2702 Monitoring Extracellular Vesicles Surface Glyco-Properties Using Fluorescent Lectins And Nanoparticle Tracking Analysis 250136 15 Open Biology United Kingdom Royal Society Publishing 2025