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Purification Analysis, Intracellular Tracking, and Colocalization of Extracellular Vesicles Using Atomic Force and 3D Single-Molecule Localization Microscopy
[Image: see text] Extracellular vesicles (EVs) play a key role in cell–cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10100414/ https://www.ncbi.nlm.nih.gov/pubmed/37002540 http://dx.doi.org/10.1021/acs.analchem.3c00144 |
Sumario: | [Image: see text] Extracellular vesicles (EVs) play a key role in cell–cell communication and thus have great potential to be utilized as therapeutic agents and diagnostic tools. In this study, we implemented single-molecule microscopy techniques as a toolbox for a comprehensive characterization as well as measurement of the cellular uptake of HEK293T cell-derived EVs (eGFP-labeled) in HeLa cells. A combination of fluorescence and atomic force microscopy revealed a fraction of 68% fluorescently labeled EVs with an average size of ∼45 nm. Two-color single-molecule fluorescence microscopy analysis elucidated the 3D dynamics of EVs entering HeLa cells. 3D colocalization analysis of two-color direct stochastic optical reconstruction microscopy (dSTORM) images revealed that 25% of EVs that experienced uptake colocalized with transferrin, which has been linked to early recycling of endosomes and clathrin-mediated endocytosis. The localization analysis was combined with stepwise photobleaching, providing a comparison of protein aggregation outside and inside the cells. |
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