Ultrafree spin filters

MVs were collected from cultures using a previously reported method [35 (link)]. In brief, each strain was cultured in 2-L marked flasks with 500 ml of TSB. Unless otherwise stated, cultures were incubated for 48 h at 15 °C in an orbital shaker (innova® 44, New Brunswick Scientific) at 180 rpm. Flasks were inoculated with 1% of pre-inoculum from a 50-ml culture of TSB grown overnight at 15 °C. MVs were isolated when cultures of each strain reached an OD₆₀₀ = 2.2. For that purpose, cells were pelleted by centrifugation at 10,000 rpm for 30 min at 4 °C, and the supernatant was filtered through 0.45-μm pore-size filters to remove remaining bacterial cells. MVs were obtained by centrifugation at 44,000×g for 1 h at 4 °C in an Avanti® J-20 XP centrifuge (Beckman Coulter, Inc). Pelleted MVs were resuspended in 50 ml of Dulbecco’s phosphate-buffered saline (PBS, Gibco, Life Technologies) and filtered through 0.22-μm pore-size Ultrafree spin filters (Millipore). After that, MVs were pelleted again and resuspended in a minimal volume of PBS.

A. baumannii AB41 and Pseudomonas PAO1 naturally secrete MVs into media. MVs from both strains were collected from broth cultures (500 ml) in the late log phase using an adaptation of the method described by McBroom and coworkers [23 (link)]. The cells were pelleted by centrifugation at 10,000 × g for 10 min at 4°C, and the supernatant was filtered through 0.45-μm-pore-size filters to remove remaining bacterial cells. MVs were obtained by centrifugation at 40,000 × g for 1 h at 4°C in an Avanti J-20 XP centrifuge (Beckman Coulter, Inc.). Pelleted vesicles were resuspended in 50 ml of 50 mM HEPES pH 6.8 (Sigma) and filtered through 0.22-μm-pore-size Ultrafree spin filters (Millipore). Vesicles were again pelleted and finally resuspended in an adequate volume of 50 mM HEPES, pH 6.8 (Sigma). MVs from N. gonorrhoeae were collected from confluent solid cultures grown on CHOC plates. Cells and MVs from 20 agar plates were resuspended in 15 ml of Ringer ¼ (Sigma) per plate and from this moment the MVs were obtained as described for liquid media cultures.
For proteomic studies, MVs from N. gonorrhoeae were further purified by ultracentrifugation in OptiPrep gradients as described by Pérez-Cruz et al. [22 (link)].

Shewanella vesiculosa M7^T naturally secrete MVs into media. MVs were collected from the 500ml TSB cultures at different times-points (12, 18, 24, 48, 72 and 96h) using an adaption of the method described by McBroom et al. (2006) (link). Cells were pelleted by centrifugation at 10,000×g for 30min at 4°C, and the supernatant was filtered through 0.45-μm pore-size filters to remove the remaining bacterial cells. MVs were obtained by centrifugation at 44,000×g for 1h at 4°C in an Avanti^® J-20 XP centrifuge (Beckman Coulter, Inc). The pelleted MVs were then resuspended in 50ml of Dulbecco’s phosphate-buffered saline (DPBS, Gibco, Life Technologies) and filtered through 0.22-μm pore-size Ultrafree spin filters (Millipore). Finally, the MVs were pelleted again at 44,000×g for 1h at 4°C and resuspended in a minimal volume of DPBS.