Protocol to isolate endosomal and small extracellular vesicles from cultured cells through ultracentrifugation.

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.1.