Isolation and Characterization of Exosome-enriched Extracellular Vesicles from Dengue Virus type 2-infected Mammalian Cells.

Exosome-enriched EVs are vesicles released from various cell types into extracellular fluids such as plasma, urine, saliva, cell culture media, and other body fluids. Their diameters range from 30 nm to 150 nm. They have been implicated in different cellular processes, including cellular communications and immune modulation during infection. Based on these effects, they have been used to study the pathogenesis of several viruses. It is challenging to isolate Exosome-enriched EVs from virus-infected cell supernatants using current purification techniques. This is largely due to the overlap in size between certain viruses and Exosome-enriched EVs. Here, we aim to purify Exosome-enriched EVs from Dengue type 2-infected cell culture supernatant. Exosome-enriched EVs were isolated using a commercial exosome isolation kit. For the isolated Exosome-enriched EVs in this protocol, no infectious virus was detected by plaque assay under the conditions tested. The purified exosome-enriched EVs were characterized using western blotting for tetraspanins, nanoparticle tracking analysis, and transmission electron microscopy for size confirmation. We demonstrated that our exosome-enriched EVs are intact and within the normal exosome diameter range of 30-150 nm. Overall, our protocol provides an inexpensive method for purifying exosome-enriched EVs from virus-infected cell culture supernatant, which can be used for downstream functional assays.