24 well culture inserts

CaCo-2 cells were seeded in 24-well culture inserts with 0.4 µm pore size (Corning Inc., Kennebunk ME, USA). Cells were grown for 3–5 days. After 30 min pre-incubation with the respective inhibitor, 100 pM TcdB were added, and cells were further incubated at 37 °C. Inhibitors were applied from the basolateral, and TcdB was applied from the apical side of the filter insert. For control, cells were left untreated or were treated with DMSO as solvent control for inhibitors. TEER was measured every hour with the EVOM2 apparatus using a STX2 electrode (World Precision Instruments Inc., Sarasota, FL, USA). Raw resistance data were normalized to 0 h time point.

The reconstructed human epidermal keratinization model (LabCyte EPI-Kit, Japan Tissue Engineering Co., Ltd. Aichi, Japan) was used according to the manufacturer’s instructions. Briefly, foreskin-derived human neonatal KCs were seeded in 24-well culture inserts (Corning Co., Ltd., Corning, NY, USA) with culture media provided by the manufacturer (assay medium; Japan Tissue Engineering Co., Ltd.) at 37 °C in a 5% CO₂ atmosphere. Twenty-four h later, the medium was removed and the KCs were air-lifted (Day 1). After the air-lift, the KCs were cultured using assay medium supplemented with 25 μg/ml vitamin C until day 7. The medium was exchanged every other day and KCs were collected at 1, 2, 5 and 7 day(s) of culture.

The human intestinal epithelial cells (Caco2 cell line) were cultured in RPMI 1640 medium (Sigma, Saint Louis, MO, USA) with 10% fetal bovine serum (Gibco, Brooklyn, NY, USA) at 37 °C in a humidified atmosphere of 5% CO₂. Caco2 cells (2 × 10⁴) were seeded on 24-well culture inserts (0.4 μm pore size; Corning, NY, USA) and grown to confluence. Thereafter, the cells were stimulated in the absence or presence of recombinant human IL-6 (R&D Systems, Minneapolis, MN, USA) or IFN-γ (R&D Systems) in the basolateral chamber up to 72 h later.
Electrical resistance across the stratified epithelium was measured using a Millicell-ERS-2 instrument (Millipore, Bedford, MA, USA) with “chopstick” electrodes, as described previously [19 (link),20 (link)]. The value obtained from a blank insert was subtracted to give the net resistance, which was then multiplied by the membrane area to give the resistance in area-corrected units (Ω·cm²). The values of transepithelial electrical resistance were recorded in a time-dependent manner after stimulation.