0.4 μm polyester membrane trans well

Non-transformed IPEC-J2 cell line (DSMZ, Braunschweig, Germany) was cultured in Dulbecco’s modified Eagle medium (DMEM:Ham’s F-12 [1:1]) supplemented with 5% fetal bovine serum (FBS), 1% insulin-transferrin-selenium-X (ITS-X) and antibiotics (all from Invitrogen, Grand Island, USA) in an incubator with atmosphere of 5% CO₂ at 37°C. To examine the effect of B. subtilis in the process of barrier formation, 5×10⁵ cells were cultured on 6-well plate for 3 days until reaching the confluence. For the electrical resistance experiment, the cells were grown on 0.4-μm polyester membrane trans-well (Corning, New York, USA) at a density of 5×10⁴ cells/well for 3 days.

IPEC-J2 cells were grown on 0.4-μm polyester membrane trans-well (Corning) until reaching the confluence of monolayer and treated with B. subtilis or LTA-BS for 1 h. And then, during barrier formation, transepithelial electrical resistance (TEER) was measured to examine the changes in integrity between the apical and basolateral side of the cells using an epithelial voltohm meter (EVOM; World Precision Instruments, Sarasota, USA). The resistance of media as a control was subtracted from each experimental value.