http://creativecommons.org/licenses/by/4.0/legalcode eng extracellular vesicles, fluorescence nanoparticle tracking analysis, ultracentrifugation, size exclusion chromatography, filtration, lectins 2025 info:eu-repo/semantics/article application/pdf 1439168 bytes Royal Society Publishing 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. Monitoring extracellular vesicle surface glyco-properties using fluorescent lectins and nanoparticle tracking analysis Monitoring Extracellular Vesicles Surface Glyco-Properties Using Fluorescent Lectins And Nanoparticle Tracking Analysis volume: 15 Mitić, Ninoslav Janjić, Filip Danilović-Luković, Jelena Goč, Sanja Janković, Tamara Janković, Miroslava https://phaidrabg.bg.ac.rs/o:37143 doi:https://doi.org/10.1098/rsob.250136