Millicell ers voltohmmeter

Cells were treated as previously described. After treatment, cells were washed with sterile PBS and TEER values were registered using a Millicell-ERS Volt-Ohm meter (MilliporeSigma, Burlington, MA, USA) according to the manufacturer’s instructions. In brief, electrodes were equilibrated for two hours in complete medium. The washing step was carried out to remove probiotics adhered to the cell monolayer that could alter the TEER measurements. The measures were repeated three times for each well. The results are obtained from the three independent experiments.

Human ME180 endocervical (ATCC no. HTB-33) and T84 colonic (ATCC no. CCL-248) epithelial cells were maintained in RPMI 1640 or Dulbecco’s modified Eagle’s medium (DMEM):Ham F12 (1:1) (Sigma-Aldrich, St. Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin/streptomycin mixture (100 units penicillin and 0.1 mg streptomycin/mL; Sigma-Aldrich) at 37°C, 5% CO₂ in saturated humidity. Cells were seeded onto 96 tissue-culture-treated well-plates (Thermo, United States) or 6 × 10⁴ cells/transwell (6.5-mm diameter and 3-μm pore size; Corning, NY) and cultured for about 10 days until transepithelial electrical resistance (TEER) reached 3000 Ω/cm². TEER was measured using a Millicell ERS Volt-Ohm meter (MilliporeSigma, Burlington, MA). The culture medium was replaced with an antibiotic-free medium the day before the experiment.

Cells were plated at a cell density of 1 × 10⁶ cells/cm² in serum-free media into 24-well Transwell inserts (0.4 μm pore size, 0.33 cm² surface area, Corning, Corning, NY, USA, Catalog #3470) coated with 100 μL of 6.6 μg/mL (2 μg/cm²) human fibronectin diluted in DPBS at room temperature. Approximately 200 μL of the cell suspension described above was added into the apical chamber and 800 μL of medium was added into the basal chamber. Cells were left undisturbed at 35 °C in a humidified 5% CO₂ incubator for 2 days before resistance measurements began. Cells were visually assessed every day after, and measurements were taken every 2 days. Resistance measurements were obtained using a Millicell-ERS Volt-Ohm Meter (MilliporeSigma, Burlington, MA, USA, Catalog #MERS00001) and STX2 chopstick electrodes (World Precision Instruments, Sarasota, FL, USA). Raw measurements were normalized by subtracting the resistance of blank fibronectin-coated inserts without cells from inserts containing cells. Transepithelial electrical resistance (TEER) was calculated by multiplying the normalized resistance measurements by the surface area of the insert.