TIRF Microscopy as a Tool to Determine Exosome Composition
Exosomes are extracellular vesicles (EVs) containing different biomolecules with biological activity, such as proteins, miRNA, long noncoding RNA, and DNA. EVs are efficient platforms for intercellular communication, especially during immune responses, bu
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TIRF Microscopy as a Tool to Determine Exosome Composition Noa B. Martı´n-Co´freces, Daniel Torralba, Marta Lozano-Prieto, Nieves Ferna´ndez-Gallego, and Francisco Sa´nchez-Madrid Abstract Exosomes are extracellular vesicles (EVs) containing different biomolecules with biological activity, such as proteins, miRNA, long noncoding RNA, and DNA. EVs are efficient platforms for intercellular communication, especially during immune responses, but also in some pathological contexts, such as tumor cell growth. The precise assessment of EV content is relevant for the selection of specific vesicles with specialized biological activities, whose content is hardly visualized due to their small size. We describe herein a protocol for the determination of the content of individual EVs through microscopy imaging and user-friendly analysis using TIRF microscopy. Keywords Extracellular vesicles, Exosomes, Cell-to-cell communication, T cells, Immune synapse, Microscopy, TIRF
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Introduction Extracellular vesicles (EVs) are lipid bilayer-delimited particles released from cells. EVs have emerged as a mode of cell-cell communication and intercellular transfer of materials, and, thus, EVs are now considered as important mediators of cell signaling [1, 2]. They have been found to be released by a wide variety of cell types, such as B and T cells, dendritic cells, platelets, cancer cells, and, even, protozoan parasites [3, 4]. EVs comprise different subtypes of particles categorized by their size, origin, and composition. Briefly, EVs are classified into exosomes when EVs size ranges from 40 to 200 nm, are produced in multivesicular bodies, and contain specific proteins located in the endosome, such as tetraspanins; into microvesicles, which are larger in size, ranging from 150 to 1000 nm, and are generated through direct budding at the plasma membrane; and apoptotic bodies, which are produced as consequence of membrane blebbing during apoptosis and are larger than 1 μm [2, 3]. Among them, exosomes have been
Sa´nchez-Madrid and Martı´n-Co´freces contributed equally to this work.
Noa B. Martı´n-Co´freces et al.
extensively studied in the last decade. In the context of the immune system, immune modulatory properties of exosomes have been reported as well as exosome transfer during immune synapse [5, 6]. The immunological activities of exosomes affect both innate and adaptive immunity, including antigen presentation, T-cell activation, or immune suppression. Furthermore, exosomes are the target of many studies as biomarkers for diagnosis and prognosis of different diseases, such inflammatory, autoimmune disorders, and cancer. Exosomes are present and stable in most body fluids, and they show a great potential as a liquid biopsy tool, which constitutes a convenient and noninvasive approach to diagnosis [7, 8]. However, EV’s small size, especially in the case of exosomes, makes it difficult to precisely characterize them. Indeed, the small size of exosomes may involve the use of very precise and specific controls to avoid confusion between
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