2.6. Transcytosis, Transepithelial Electrical Resistance (TEER) and Paracellular Permeability

In order to determine whether BL23 and LGG had the ability to cross through the Caco-2 or T84 monolayer, transcytosis assays were performed as described before in [41,42,43], with slight modifications. Briefly, Caco-2 and T84 cells were seeded on transwell culture inserts (as explained above) and the plates were incubated for 10 and 17 days, respectively. During this period, the transepithelial electrical resistance (TEER) was determined with a specialized electrode (Millipore). The experiment started when reaching a minimum TEER value of 200 Ω.cm² for the Caco-2 and 1000 Ω.cm² for the T84 monolayers. To start the experiments, the culture medium was replaced by eukaryotic growth medium without antibiotics. Bacteria were inoculated at the apical compartment of the insert at a MOI of 10³, where LY (50 μmol/L) was added as control of the paracellular permeability. After 5 to 24 h of incubation (37 °C, 95% humidity, 5% CO₂), the concentration of bacteria in the basolateral chamber was determined by inoculating serial dilutions on MRS-agar plates. The concentration of LY in the basolateral compartment was determined by measuring the fluorescence emission at 530 nm. When appropriate, the effect of the inhibitors ML-7 and dynasore was tested, preincubating the monolayer for 30 min before the addition of bacteria. IFN-γ was also tested, with 48 h preincubation in the basolateral compartment.