20.500.12556/RUL-183558 Freeze-fracture electron microscopy for extracellular vesicle analysis Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs. extracellular vesicles electron microscopy urothelial cells cancer zunajcelični vezikli elektronska mikroskopija urotelijske celice true false true Angleški jezik Slovenski jezik Članek v reviji 2026-06-15 09:54:53 2026-06-15 09:55:01 2026-07-09 09:33:53 0000-00-00 00:00:00 2022 0 0 13 str. Vol. 187 2022 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 616.6 1940-087X 10.3791/63550 125862659 RAZ_Resnik_Natasa_2022.pdf RAZ_Resnik_Natasa_2022.pdf 1 C30A9C66471CC1439BB0912CAC0ABA99 707d27793c761f73edecc4fe774f7ce3f19e7cc0a835c1198eef4924c77c31b8 bcdcab80-688f-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=235623 https://www.jove.com/t/63550/freeze-fracture-electron-microscopy-for-extracellular-vesicle-analysis 1 7e7f3097-688f-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=235622 Medicinska fakulteta 0 0 0