Polyethylene terephthalate

HBMEC/ciβ were routinely grown on type-I collagen-coated dishes in CSC-cbR at 33°C with 5% CO₂/95% air. They were seeded (day 0) at 1.0 × 10⁵ or 4.0 × 10⁵ cells/mL onto a dish or a membrane filter of an insert culture system (polyethylene terephthalate, 0.4 μm high-density pores, and 0.3 cm², BD Falcon, Franklin Lakes, NJ, USA), respectively. At day three, the medium was changed to either the same or a differently supplemented medium, depending on the experiments (for example, the medium was changed from CSC-cbR to CSC-HC at day three). Then, the cells were continuously cultured for 12 days, during which a medium change was conducted every other day. All functional or gene expression analyses were performed on day 12. The culture schedule is also provided in Additional file 1: Figure S1.

The CDI assay followed a previously described protocol (Aoki et al., 2005 (link)). Polyethylene terephthalate (PET) track-etched membrane inserts (23 mm) of 0.4 μm pore size (Falcon, Corning, NY) were placed in six-well plates to create upper and lower culture wells. Overnight cultures of PA2 and non-pathogenic E. coli strains were diluted to an OD₆₀₀ of 0.05. Diluted PA2 (3.2 mL) and non-pathogenic E. coli (2.5 mL) were added to the bottom and top chambers, respectively. Plates were incubated at 37°C with shaking at 130 rpm for 6 h. Both top and bottom samples were 10-fold serially diluted in PBS and 100 μL aliquots were plated onto SMAC plates to ensure no cross contamination occurred. After harvesting the cells and treating them with PMB for 5 min at 37°C, samples from the bottom chamber were centrifuged at 10,000 × g for 1 min, and supernatants were stored for immediate use or at −80°C. Stx2a levels were evaluated by R-ELISA.

Measurements were performed using a pH-sensitive fluorescent probe, BCECF/AM (2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester; Life Technologies) and the fluorescence intensity was recorded at excitation wavelengths of 500 and 440 nm and at emission wavelength of 535 nm by a computer controlled spectrofluorometer (Cary Eclipse Varian). Briefly, bronchial epithelial cells grown on permeable supports in polyethylene terephthalate (BD Falcon) at ALC conditions, and loaded with BCECF, were mounted in a cuvette which allowed independent perfusion of the apical and basolateral sides. The solution contained (in mM): 115 NaCl, 5 KCl, 25 NaHCO₃, 1 MgCl₂, 1 CaCl₂, 10 D-glucose. Solution pH was adjusted to 7.4 by bubbling with 95% O₂ and 5% CO₂. For Cl⁻-free conditions, NaCl was substituted with sodium gluconate, CaCl₂ with 6 mM calcium gluconate, and KCl with 2.5 mM K₂SO₄. Experiments were carried out at 37 °C.
Calibration of fluorescence ratio to pH_i was performed after each experiment using nigericin (10 μM) and potassium ions (150 mM) at various external pH values varying between 6.0 and 8.0 as previously reported37 (link). The value of ∆pH_i was calculated as the difference between the maximum value reached upon the perfusion with Cl-free solution and the value obtained by averaging the last 20 data points recorded before perfusion with the Cl⁻-free solution.