2.6. Caco-2 TransEpithelial Electrical Resistance (TEER) Assay

Caco-2 cells (400 µL at a density of 3.5 × 10⁵ cells/mL) were seeded on Merck-Millipore^® Transwell^® polycarbonate cell culture inserts with a 0.4 µm filter membrane and a 0.6 cm² surface area. They were placed in Cellstar^® 24-well plates. The values of TEER were determined by measuring the potential difference between the two sides of the cell monolayer using a Millicell^® ERS-2 m (Millipore^®, Billerica, MA, USA) connected to a pair of chopstick electrodes. TEER measurements and medium changes were carried out every two days to maintain optimal culture conditions and to make sure of the complete maturity of the cell monolayer i.e., 6 days of culture in our experimental conditions.

To evaluate the influence of yeasts and ST on the epithelial cell barrier, mature cell cultures were washed with PBS prior to the addition of 400 µL of each microorganism, which was resuspended in DMEM at a final concentration of 10⁶ CFU/mL. TEER measurements were made every hour for 8 h and then once again at 24 h.

In order to evaluate the influence of Dh25 on the TEER modulation by ST, co-cultures of 24 h, 48 h, 72 h, and 96 h were made and resuspended in DMEM. A volume of 400 µL of these four co-cultures was inoculated into the monolayer cells. TEER measurements were made every hour for 16 h, and once at 24 h, in comparison with 400 µL of ST alone, at a concentration of 6.3 log CFU/mL.

The resistance value of each individual Transwell was calculated by subtracting the resistance of a coated Transwell without cells from the total resistance (cell epithelium with or without microorganism plus filter insert). Values at 0 h were normalized to 100%. Data were presented as the average of three independent experiments.