For stage 2, further optimisations were applied to methods D and N as follows: The single AW buffer wash step in method D was increased to three washes, and the supplied elution buffer was replaced with 2 × 50 μL 10 mM Tris (pH 8.5).
The buffer was replaced with a fresh buffer containing 3.7 mM glucose for one hour before the buffer were collected (basal) and replaced with a buffer containing 16.7 mM glucose for an additional one hour (glucose stimulated).
After each time point, the plates were placed on ice, and the ligand binding buffer was replaced with ice-cold strip/stop solution.
For testing the effect of Mn++, Mg OAC 2 (5 mM) in the buffer was replaced with varying concentrations of MnCl2 (0.5, 1, 5, 7.5 mM).
After the samples had cooled to room temperature, the DTT-containing buffer was replaced with iodoacetamide-containing buffer (55 mM iodoacetamide 100 mM NH4HCO3), and the gel pieces were vortexed for 45 min at room temperature.
The blocking buffer was replaced with 100 µl of zipper-scFv G28-5 prorein or 293T cell lysates containing zipper-modified fiber proteins diluted in binding buffer (TBS with 0.05% Tween 20 and 0.05% casein).
The mucus plume was allowed to grow for 45 min, the loose mucus was removed and the thickness was measured before the apical buffer was replaced with the same buffer containing 3% DSS or 3% Dextran.
Extraction buffer was replaced with Digestion buffer (50 mM NaCl, 300 mM sucrose, 10 mM Pipes pH 6.8, 3 mM MgCl2, 0.5% Triton X-100, 1 mM PMSF, 20 units/mL RNAse inhibitor, and 200 500 units/mL RNase-free DNase) and incubated for 60 min at room temperature.
On each occasion the collected buffer was replaced with fresh buffer to maintain constant volume.
The pre-hybridization buffer was replaced with RNA probe in HB (5 ng/mL) and incubated overnight at 60°C.
