Isolation and Characterization of Outer Membrane Vesicles

To isolate OMVs, we followed a previously reported protocol, with modifications (Liu et al., 2016b (link)). Bacteria were grown in LB at 37°C with shaking (approximately to OD₆₀₀ = 1.1), prior being centrifuged 10 min at 5400 ×g at 4°C. The pellet was discarded, and the supernatant fraction was filtered (0.45 μm), ultrafiltered with Ultracel^® 100 kDa ultrafiltration disks (Amicon Bioseparations), and ultracentrifuged 3 h at 150000 ×g at 4°C (Thermo Scientific™ Sorvall™ WX, Rotor AH-629). The supernatant was discarded, and the pellet resuspended in 1 ml DPBS. OMVs were stored at -20°C until their use. We quantified OMV yield as by determining the both protein content (BCA assay) and lipid content (FM4-64 molecular probe), and normalizing by CFU/ml (McBroom et al., 2006 (link); Deatherage et al., 2009 (link)). We determined OMV size as described (Deatherage et al., 2009 (link)). Briefly, OMV size (diameter) was measured from at least 3 TEMs of 3 independent OMV extracts per strain in Adobe Photoshop using the ruler tool. Results were presented in diameter ranges of 3 nm, where 3 represents 1 to 3 nm. All OMVs larger than 60 nm were grouped in the last category (60+ nm).