Isolation and Characterization of Exosomes from R. parkeri-Infected HUVECs

After R. parkeri at a MOI of 10 or mock infection for 72 hours, 11 mL of media from HUVECs in T75 flasks was collected and filtered twice using 0.2 µm syringe filters. Ten mL of filtered media was placed in the qEV10 column (Izon, Medford, MA) for Exo isolation according to the manufacturer’s instructions. Number 7 through 10 fractions were collected and pooled as the Exo-enriched fractions, which were concentrated using 100,000 MWCO PES Vivaspin centrifugal filters (Thermo Fisher Scientific, Waltham, MA). All isolated bacterium-free Exos samples were routinely confirmed using plaque assay as we described (79 (link)). Conditioned culture media from HUVECs that were infected with R. parkeri for 72 hours were used as positive control prior to the filtration. Nanoparticle tracking analysis (NTA) was performed using a qNano Gold system (Izon, New Zealand) to determine the size and concentration of extracellular vesicle particles of each Exo sample. Exo samples (in 200 µl PBS) were stored at -80°C for further use (26 (link)).