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An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles

2021; Springer Science+Business Media; Linguagem: Inglês

10.1007/978-1-0716-1205-7_24

1940-6029

Carmen Campos‐Silva, Yaiza Cáceres‐Martell, Sheila López‐Cobo, Maria Josefa Rodríguez, Ricardo Jara, María Yáñez‐Mó, Mar Valés‐Gómez,

MicroRNA in disease regulation

Most human cells release extracellular vesicles (EVs) of different sizes and composition, containing biomolecules characteristic from the originating tissue. In consequence, when EVs derive from a cancer cell, they also contain tumor antigens. Therefore, isolating and characterizing tumor-derived EVs has attracted great interest as an invaluable source of biomarkers, both for diagnosis and stratification of cancer. In this chapter, we describe a method for flow cytometry assessment of melanoma-derived EVs which are firstly captured onto antibody-coated beads recognizing either a common EV marker, namely, a tetraspanin, or a tumor antigen like the stress-related molecules MICA or PDL1. Then, after staining with a fluorophore-conjugated antibody directed against a different protein present on the EV surface, the EV-bead complex can be visualized in a conventional flow cytometer. The technique allows detection of proteins present on EVs isolated from tissue culture supernatants of melanoma cell lines and, more importantly, directly from plasma.