Lyophilized rhil 6 or rhil 8

Lyophilized rhIL-6 or rhIL-8 (PeproTech) at 10 μg/ml final concentration was incubated with P. aeruginosa filtrates [3% (v/v) final concentration] at 37°C. At specified times, 10-μl aliquots were mixed 1:1 (v/v) in tricine buffer [200 mM tris-HCl, 40% glycerol, 2% SDS, 0.04% Coomasie blue G-250, 2% β-mercaptoethanol (pH 6.8)] and heated to 95°C for 5 min. Samples were then separated by SDS-PAGE with 16.5% tris-tricine gels. After transfer of gels onto nitrocellulose paper, the blots were blocked with 2% BSA and then immunoblotted with polyclonal human IL-6 or IL-8 antibodies (R&D Systems) diluted in PBS (with 0.1% BSA, 0.05% Tween 20). The signal was detected using donkey α-goat secondary antibody conjugated to IRDye 800 infrared fluorophore (Li-Cor Biosciences) in PBS with 0.05% Tween 20. Images of the fluorescently labeled protein bands were captured with the Odyssey imaging system (Li-Cor Biosciences) and analyzed using ImageJ software [1.47v, National Institutes of Health (NIH)]. The protein band density was quantified and normalized to the T = 0 control on the same blot.