Isolation and Characterization of Extracellular Vesicles

The culture was isolated as described by Vallejo et al. (19 (link)) with slight modifications. After 72 h of culture in the minimal medium, the supernatant was obtained using sterile Miracloth (Millipore, Billerica, MA, USA) and was concentrated via the use of an Amicon ultrafiltration system (Millipore, Billerica, MA, USA) (100-kDa cutoff), centrifuged at 15,000 × g for 15 min at 4°C, and ultracentrifuged at 60,000 rpm at 4°C for 1 h. The EVs were suspended in 500 μl of sterile nuclease-free water (Sigma-Aldrich, St. Louis, MO, USA).
The size and distribution of EVs from independent growth cultures were verified via nanoparticle tracking analysis (NTA) using a NanoSight NS300 system (Malvern Instruments, Malvern, United Kingdom) as previously described (24 (link)). The particle size, distribution, and quantification were carried out using NanoSight software (version 3.2.16).