To determine if OMVs were able to bind to BC and quench its fluorescence, the fluorescence titration of OMVs was performed based on a previous assay (Wood et al., 2004 (link)). BC was diluted in 50 mM Tris-HCl (pH 7.4) to make a final concentration of 4 μM. The BC-OMV solutions were prepared by the addition of aliquots of stock solutions of OMVs into BC solutions, of which the final concentration of OMVs ranged from 0.1 to 4 μg/ml. A 100 μl of BC-OMV solution was added into a non-binding surface (NBS) black 96 well microplate (Corning, NY, USA) in triplicate. The fluorescence intensity was read with excitation at 580 nm and emission at 620 nm (Enspire 2300, PerkinElmer, MA, USA).
The displacement of OMV-bound BC by colistin was performed as previously described (French et al., 2019 (link)) with slight modifications. Briefly, 100 μl of 50 mM Tris-HCl (pH 7.4) buffer containing 4 μM BC, 2 μg/ml OMVs and serially diluted colistin (0.02 to 200 μg/ml) was added into NBS black 96 well microplate (Corning), and the fluorescence intensity was read.
The displacement of OMV-bound BC by colistin was performed as previously described (French et al., 2019 (link)) with slight modifications. Briefly, 100 μl of 50 mM Tris-HCl (pH 7.4) buffer containing 4 μM BC, 2 μg/ml OMVs and serially diluted colistin (0.02 to 200 μg/ml) was added into NBS black 96 well microplate (Corning), and the fluorescence intensity was read.