Outer Membrane Vesicles of V. fischeri

Outer membrane vesicles (OMVs) produced by V. fischeri ES114 were prepared as previously described (61 (link), 62 (link)), with the following modification. After OMVs were separated from other extracellular products by ultracentrifugation, they were further purified by using linear 10 to 50% sucrose gradient centrifugation at 100 000 × g for 16 h at 4°C in a 90 Ti rotor (Beckman Coulter, Inc., Brea, CA). To treat squid with OMVs, 100 μg·ml⁻¹ was added to FSW and incubated with hatchling animals for 24 h (61 (link), 62 (link)). The peptidoglycan monomer (tracheal cytotoxin [TCT]) and lipid A from V. fischeri were added to FSW for 24 h at a concentration of 1 μM and 10 μg·ml⁻¹, respectively (25 (link), 26 (link)).