Purification of Bacterial-Enriched Extracellular Vesicle Samples from Feces by Density Gradient Ultracentrifugation.

Commensal microbiota has huge impact on the maintenance of human health, its dysregulation being associated with the development of a plethora of diseases. Release of bacterial extracellular vesicles (BEVs) is a fundamental mechanism of systemic microbiome influence on the host organism. Nevertheless, due to the technical challenges of isolation methods, BEV composition and functions remain poorly characterized. Hereby, we describe the up-to-date protocol for isolation of BEV-enriched samples from human feces. Fecal extracellular vesicles (EVs) are purified through the orthogonal implementation of filtration, size-exclusion chromatography (SEC), and density gradient ultracentrifugation. EVs are first separated from bacteria, flagella, and cell debris by size. In the next steps, BEVs are separated from host-derived EVs by density. The quality of vesicle preparation is estimated via immuno-TEM (transmission electron microscopy) for the presence of vesicle-like structures expressing EV markers and via NTA (nanoparticle tracking analysis) for assaying particle concentration and size. Distribution of EVs of human origin in gradient fractions is estimated using antibodies against human exosomal markers with Western blot and ExoView R100 imaging platform. The enrichment for BEVs in vesicle preparation is estimated by Western blot for the presence of bacterial OMVs (outer membrane vesicles) marker and OmpA (outer membrane protein A). Taken together, our study describes a detailed protocol for EV preparation with enrichment for BEVs from feces with a purity level suitable for bioactivity functional assays.