Quantitative Measurement of Extracellular Vesicle Content Delivery Within Acceptor Cells

Extracellular vesicles (EVs), including exosomes and microvesicles, are thought to transport bioactive molecules from donor to acceptor cells. Although EV uptake has been qualitatively assessed through subcellular imaging, EV content delivery has been rarely addressed due to a lack of adequate methods. Here we present a sensitive bulk assay to quantitatively measure EV uptake and content delivery in mammalian cell. In this assay, EVs containing a NanoLuc luciferase-tagged cargo are mixed with unlabeled acceptor cells. Cell fractionation separates membrane and cytosolic fractions, and luciferase activity is measured within each fraction to determine the percentage of cytosolic release. This assay can be used to further decipher cellular and molecular mechanisms that regulate the EV delivery process or to quantitatively test specific pairs of donor-acceptor cells.