We used P. aeruginosa non-isogenic, mucoid (ATCC 39324) and non-mucoid (ATCC 27318) strains for all studies as recently described [21 (link)]. Briefly, strains were grown overnight in Luria-Bertani medium (LB; Becton Dickson) at 37°C with shaking. The next day, 50 μL of overnight culture was added to 2 mL LB media and allowed to grow to mid-log phase, before adjusting suspensions to an optical density at 600 nm of 0.1 (1× 109 CFU/mL). Bacterial suspensions for infection were prepared by pelleting them at 450× g for 10 minutes, washing them 2× with sterile phosphate-buffer saline (PBS), followed by re-suspension of bacteria to the same volume in culture medium without antibiotics. Bacteria were further diluted from 109 to 108 CFU/mL depending on the specific experiment. Quantitative analysis of bacterial numbers was determined by 100-fold serial dilutions, before plating on LB agar plates. Plates were incubated overnight at 37°C and bacteria were enumerated the following day.
P. aeruginosa flagellin (FLA) (Invivogen, San Diego, CA) was dissolved in water at a concentration of 0.2 mg/mL and stored at −80°C. P. aeruginosa LPS (Sigma-Aldrich, St Louis, MO) was used by dissolving the lyophilized powder in water at 1 mg/mL and stored at 4°C. Both FLA and LPS were diluted in antibiotic-free culture media to their optimum concentrations as predetermined in this study.
P. aeruginosa flagellin (FLA) (Invivogen, San Diego, CA) was dissolved in water at a concentration of 0.2 mg/mL and stored at −80°C. P. aeruginosa LPS (Sigma-Aldrich, St Louis, MO) was used by dissolving the lyophilized powder in water at 1 mg/mL and stored at 4°C. Both FLA and LPS were diluted in antibiotic-free culture media to their optimum concentrations as predetermined in this study.
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