Extracellular Vesicle Uptake Assay: A Method to Visualize and Quantify Cellular Uptake of Extracellular Vesicles Using 3D Fluorescence Imaging Technique

To facilitate cellular communication, cells secrete extracellular vesicles, EVs - nano-sized structures enclosed by a lipid membrane. EVs carry various biological cargoes, including macromolecules and metabolites, for delivering them to the recipient cells.

To visualize EV uptake by cells using confocal microscopy, first, take freshly isolated EVs. These EVs are pre-labeled with antibodies bound to a fluorescent probe for subsequent easy visualization.

Add an adequate volume of fluorescently-labeled EVs to an adherent recipient cell culture. Incubate for the desired period. During incubation, a sub-population of the labeled EVs superficially attach to the cell surface, while other EVs get internalized.

Add a different fluorescent dye to label the cell cytoplasm, helping distinguish internalized EVs from those adhered to the recipient cell. Place the cells under a confocal microscope and image at different focal planes along the z-axis, acquiring a series of optical sections across the sample thickness.

Compile this collection of optical sections - a z-stack - to form a high-resolution, three-dimensional sample image. Apply virtual rendering - a post-imaging process - to reconstruct the cell surfaces.

To differentiate superficially adhered from internalized EVs, render them as dots of different hues. Calculate the cell volume and number of EVs internalized by them, quantifying EV uptake per cell.