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Freeze-fracture electron microscopy for extracellular vesicle analysis [Elektronski vir]Resnik, Nataša ...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.Source: Journal of visualized experiments [Elektronski vir]. - ISSN 1940-087X (Vol. 187, 2022, str. 1-13)Type of material - e-article ; adult, seriousPublish date - 2022Language - englishCOBISS.SI-ID - 125862659
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Database name | Field | Year |
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Links to authors' personal bibliographies | Links to information on researchers in the SICRIS system |
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Resnik, Nataša | 28393 |
Romih, Rok | 11654 |
Erdani-Kreft, Mateja | 15873 |
Hudoklin, Samo, biolog | 25780 |
Source: Personal bibliographies
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