Endocytosis Assays Using Cleavable Fluorescent Dyes

Endocytosis mediates the entry of surface and extracellular cargoes into the cell. In this chapter, we describe assays to quantitively measure the endocytosis of both soluble and transmembrane cargo proteins, taking advantage of cleavable fluorescent dyes labeling cargo proteins or antibodies recognizing cargo proteins. After removing surface-bound fluorescent dye, internalized cargoes are measured using confocal imaging and flow cytometry. We also describe strategies to determine the role of clathrin-mediated endocytosis (CME) in the internalization of a cargo by using a small molecule inhibitor of CME and knockout (KO) of the AAGAB gene, which encodes an essential regulator of CME.