Domingues, Maria NolascoPalhinhas, LuísPereira, PauloFerreira, João Vasco2026-06-262026-06-262026-06-192666-1667PURE: 163159536PURE UUID: 9553fffc-ce64-4f7d-a6ed-dae9359b46b8Scopus: 105037400461ORCID: /0000-0002-9908-2290/work/218920305http://hdl.handle.net/10362/204134Publisher Copyright: © 2026 The Authors.Here, we present a protocol to isolate endosomal fractions using sucrose-density gradient ultracentrifugation and to recover small extracellular vesicles (sEVs), enriched in exosomes, using sequential ultracentrifugation from mammalian cell lines. This combined approach enables the separation and analysis of early endosome (EE), late endosome (LE), and sEV fractions. We provide detailed procedures for cell culture preparation, conditioned media collection, differential centrifugation, gradient layering, and fraction purification, facilitating downstream characterization and functional assays. For complete details on the use and execution of this protocol, please refer to Ferreira et al.2669980engGeneral NeuroscienceGeneral Immunology and MicrobiologyGeneral Biochemistry,Genetics and Molecular Biologyjournal article10.1016/j.xpro.2026.104531https://www.scopus.com/pages/publications/105037400461