a. Schematic showing biotin/streptavidin linking S. aureus to gold grids that were placed on the airway surface. After removal, bacteria were exposed to fluorescent live/dead stain (SYTO 9/propidium iodide), imaged, and counted.
b. Scanning electron photomicrographs of bacteria-coated grid (top), grid bar (middle), and individual bacteria (bottom).
c. Image of bacteria-coated grid (green=live, red=dead) after placement for 5min on tracheal surface of 1-month-old, wild-type pig. Bottom shows percentage of bacteria that were dead after immersion in saline, water, 70% ethanol, or placement on tracheal surface. n=3 each. Here and elsewhere, bars are SEM.
d. S. aureus-coated grids were placed on tracheal surface of newborn CF and non-CF pigs for indicated times. Data are percentage dead bacteria. Each set of 3 time-points is from a single animal. Letters (a–f) indicate littermates; there is no 1min time point for CF pig in litter c due to experimental error. For each pig, 2–3 grids were used at each time point, 5–16 fields were counted per grid, each field contained ~100–1000 bacteria (), and data from each field were averaged. Operators were blinded to genotype. CF was different from non-CF at all time points, P<0.01.
e. S. aureus-coated grids were placed for 30sec on tracheal surface before and ~5min after methacholine stimulated secretion. n=6/genotype. *P<0.02. For each genotype, data with and without methacholine do not differ significantly.
f. ASL was removed from methacholine-stimulated pigs, bacteria (1×10⁶ CFU/ml) were incubated in 1µl ASL for 10min. in a micro CFU assay, and bacteria were counted by dilution plating. n=6/genotype. *P<0.05.
g. S. aureus-coated grids were placed on surface of primary epithelial cultures for indicated times, and percentage dead bacteria was determined. Each set of data points represents mean from epithelia from a different animal. CF was different from non-CF at all time points, P<0.001.
h. P. aeruginosa-coated grids were placed on surface of cultured epithelia for 5min. n=5 cultures from different pigs per genotype. *P<0.001.
i. S. aureus in 100nl H₂O were applied to apical surface of cultured epithelia. Apical surface was washed 24hr later and bacteria counted by dilution plating. Data are % of epithelia that showed no bacterial growth. n=18/genotype. *P<0.005, Fisher's exact test.

a. Lysozyme concentration was measured with lysoplates; n=8/genotype. Quantitative western blots assayed lactoferrin, PLUNC, and SP-A; data are relative intensity of blots, n=12/genotype.
b. 60µl isotonic xylitol was added to apical surface of airway cultures (diluting ASL ~100:1). 3min later, two S. aureus-coated grids were placed on epithelial surface for 1min, then removed and counted. n=6 epithelia/genotype, each from different pigs.
c. ASL was removed from cultured epithelia by washing with 100µl H₂O. ASL was incubated with S. aureus (3.3×10³ CFU/ml) for 60min, and micro CFU assays were used to measure antimicrobial activity. n=12/genotype. See also .
d. Methacholine-stimulated ASL was diluted 1:100 in H₂O, incubated with S. aureus (1×10⁶ CFU/ml) for 60min, and CFU assays were used to measure antimicrobial activity. n=5/genotype.
e. Radial diffusion assays were used to measure antimicrobial activity of ASL collected from epithelial cultures. n=6/genotype.

a. ASL was collected under basal conditions from tracheal surface using Parafilm-coated paper; Na⁺ and K⁺ concentrations were measured as described in Methods (). n=8 non-CF and 6 CF.
b. ASL Na⁺ and K⁺ concentrations in methacholine-stimulated ASL. n=16 non-CF and 14 CF pigs. *P<0.05.
c. ASL pH measured in vivo using pH-sensitive planar optical probe placed on tracheal surface. n=6 non-CF and 7 CF; littermates were used with one extra CF. *P<0.05. Studies were done with animals in 5% CO₂; therefore, ASL CO₂ concentration was likely >5% due to CO₂ production by the pigs.
d. Methacholine-stimulated ASL was removed, and pH was measured with a micro-optical pH probe. pH was measured 10min after removal in ambient CO₂, which likely increased pH values compared to in vivo. n=10 pigs/genotype; littermates were used. *P<0.0005.
e. ASL pH measured in cultured airway epithelia using fluorescent pH indicator. N=5epithelia/genotype, each from a different pig. *P<0.01. Calculated HCO₃⁻ concentrations using measured pH, the 5% CO₂ concentration, and Henderson-Hasselbalch equation were non-CF 28.1±4.2mM (n=8) and CF 13.1±2.4mM (n=8), p=0.007.

a. Methacholine-stimulated ASL was removed from non-CF pigs, pH was adjusted with HCl, and antimicrobial activity was measured with S. aureus-coated grids. n=6. See .
b. Effect of pH on antimicrobial activity of 1.25mg/ml lysozyme and 4mg/ml lactoferrin in S. aureus luminescence assay. Data are relative luminescence compared to control at 30min. for lysozyme and 60min. for lactoferrin (). Similar results were obtained with E. coli ().
c. Tracheas of non-CF pigs were exposed to 0% or 15% CO₂ in vivo. pH was measured with pH-sensitive planar electrode. n=6/genotype. Bacterial killing was measured with S. aureus-coated grids placed on surface for 30sec. n=4/genotype. *P<0.05.
d. NaHCO₃ or NaCl (50µl, 100mM) was aerosolized onto airway surface of CF pigs. pH (n=6/genotype) and bacterial killing (n=5/genotype) were measured as in 4c. *P<0.05.