Abstract
Detection of tumor necrosis factor-alpha (TNF-α) is usually performed in cell cultured medium or body fluids via measurement of its soluble extracellular form. However, depending on cellular condition, TNF-α might be transported through extracellular vesicles (EV) from donor cells to recipient cells. EV are small membrane-delimited structures (∼50 nm to 10 μm) that are spontaneously released from multiple cell types. In cancer, EV arise as important mediators in intercellular communication, and their molecular content may support tumor progression. This chapter describes methods to identify protein content in EV released from the tumor cell cultures. Through this protocol, we show first how to purify EV from in vitro cell culture by using differential centrifugation technique and then we demonstrate how to identify both membrane and soluble TNF-α forms in EV by Western blotting.
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Acknowledgements
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-304565/2016-4). TSB was supported by Ministério da Saúde/INCA; PSS is supported by Coordenação de Aperfeiçoamento de pessoal de Nível Superior (PNPD/CAPES).
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Berguetti, T.S., Maia, R.C., de Souza, P.S. (2021). Detection of TNF-α Protein in Extracellular Vesicles Derived from Tumor Cells by Western Blotting. In: Bayry, J. (eds) The TNF Superfamily. Methods in Molecular Biology, vol 2248. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1130-2_19
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DOI: https://doi.org/10.1007/978-1-0716-1130-2_19
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1129-6
Online ISBN: 978-1-0716-1130-2
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