Evom2 apparatus

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.

Caco-2-cells (125,000/well) were seeded in 24-well cell culture inserts with a 0.4 µm pore size (for TcdB: Millicell Hanging Cell Culture Insert, Millipore by Merck KGaA, Darmstadt, Germany; for TcdA and TcdA + TcdB: BRAND inserts, BRAND GMBH + CO KG, Wertheim, Germany) and grown for four days. TcdA (200 pM) and/or TcdB (100 pM) as well as α-def-6 (6 µM) were added apically. H₂O was included in the negative and positive controls as a solvent control for α-def-6. TEER was measured every 0.5 h with the EVOM² apparatus connected to the STX2 electrode (both World Precision Instruments Inc., Sarasota, FL, USA). Raw resistance data were normalized to time point t = 0 h.