Biochemical Reconstitution of Ca²⁺-Dependent Exosome Secretion in Permeabilized Mammalian Cells

Exosomes are a subpopulation of the heterogenous pool of extracellular vesicles that are secreted to the extracellular space. Exosomes have been purported to play a role in intercellular communication and have demonstrated utility as biomarkers for a variety of diseases. Despite broad interest in exosome biology, the conditions that regulate their secretion are incompletely understood. The goal of this procedure is to biochemically reconstitute exosome secretion in Streptolysin O (SLO)-permeabilized mammalian cells. This protocol describes the reconstitution of lyophilized SLO, preparation of cytosol and SLO-permeabilized cells, assembly of the biochemical reconstitution reaction, and quantification of exosome secretion using a sensitive luminescence-based assay. This biochemical reconstitution reaction can be utilized to characterize the molecular mechanisms by which different gene products regulate exosome secretion. Key features This protocol establishes a functional in vitro system to reconstitute exosome secretion in permeabilized mammalian cells upon addition of cytosol, ATP, GTP, and calcium (Ca²⁺).