Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

Abstract

The secretion of extracellular vesicles by cestodes is crucial for enabling cellularcommunication not only among parasites but also with host tissues. In particular,small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens,which are critical in host immunomodulation and parasite survival. This article presentsa step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcusgranulosus and analyzes their uptake by dendritic cells obtained from murine bonemarrow, which acquire adhesion and antigen presentation capacity during theirmaturation after one week of in vitro culture. This article provides comprehensiveinformation for generating, purifying, and quantifying sEVs using ultracentrifugationalongside parallel analyses of dynamic light scattering and transmission electronmicroscopy. Additionally, a detailed experimental protocol is outlined for isolating andcultivating mouse bone marrow cells and driving their differentiation into dendriticcells using Flt3L. These dendritic cells can present antigens to naïve T cells, therebymodulating the type of immune response in vivo. Thus, alternative protocols, includingconfocal microscopy and flow cytometry analysis, are proposed to check the acquiredmaturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally,it is worth noting that the described protocol can be applied as a whole or in individualparts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bonemarrow-derived dendritic cell cultures, and perform uptake assays with these cells.