Tobramycin was used at 1,000 ��g/ml, the peak concentration measured in the BAL isolated from the lungs of patients selleck Bortezomib with CF (21). TABLE 1. DRUGS AND COMPOUNDS USED IN THIS STUDY Cell Lines and Cell Culture Human bronchial epithelial cells (CFBE41o?) homozygous for the ��F508-CFTR mutation and stably overexpressing ��F508-CFTR (hereafter called CFBE cells) (32, 33) were maintained in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 2 ��g/ml puromycin, 5 ��g/ml plasmocin, 50 U/ml penicillin, and 50 ��g/ml streptomycin in a 5% CO2�C95% air incubator at 37��C. We have previously reported that these growth culture conditions lead to an extracellular iron concentration of approximately 0.1 ��M (20). Antibiotics were removed immediately before experiments.

CFBE cells were seeded on 40-mm diameter glass coverslips (Bioptechs, Butler, PA) at 2 �� 106 cells/coverslip and grown at 37��C for 8 to 10 days to establish confluent monolayers. In some experiments, CFBE cells were seeded at 1 �� 106 cells on 24-mm permeable filter inserts (Snapwell; Corning Costar, Kennebunk, ME) and grown in air�Cliquid interface culture at 37��C for 8 to 10 days to establish confluent monolayers. In a previous study we found that P. aeruginosa biofilm formation was similar on parental CFBE41o? cells, on the CFBE cells stably overexpressing ��F508-CFTR, and on human airway epithelial cells that produce mucus (20). Bacterial Strains P. aeruginosa strain PAO1 carrying the pSMC21 plasmid was grown in lysogeny broth (LB) overnight.

This plasmid constitutively expresses green fluorescent protein (GFP), and is stable in the absence of antibiotic selection (34, 35). As described in detail previously (20), for co-culture studies with CFBE cells, overnight cultures of P. aeruginosa were washed, resuspended in microscopy medium (see below), and used at a multiplicity of infection (M.O.I.) of 25. Flow Chamber Imaging Experiments GFP-labeled P. aeruginosa biofilms were grown on the apical side of live, polarized CFBE cells in a FCS2 flow chamber (Bioptechs) as described previously (20). Briefly, polarized and confluent CFBE cells grown on glass coverslips were placed in the flow chamber and perfused with a modified cell growth medium (MEM without phenol red, 2 mM L-glutamine), hereafter referred to as ��microscopy medium.

�� Bacteria were injected on the apical side of the cells and allowed to attach, without flow, for 2 hours. The flow was subsequently started and maintained throughout the experiment at a rate of 20 ml/hour. Microscopic observations were performed on a Nikon LiveScan Swept Field confocal microscope (Tokyo, Japan) or on an Olympus IX-70 inverted microscope (Tokyo, Japan). Digital Drug_discovery images were acquired with the Nikon Software’s Suite or with the OpenLab 4.0.3 software package (Improvision, Inc.