Cell culture, TNF-α stimulation, and transepithelial electrical resistance assays

Caco2 and T84 cell can form monolayers in plates, and are used widely as colon epithelium models to mimic the epithelium of the human colon mucosa.³ Caco2 and T84 cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). For more details of cell culture, see Supplemental Digital Content 1.

Following establishment of monolayer of Caco2 and T84 cells at about 12 days after seeding, TNF-α (Sigma, St Louis, MO, USA) was added to the medium at three different concentrations (5 ng/ml, 10 ng/ml, and 20 ng/ml) to mimic different disease activity in CD. Another group of cells had double distilled water added as a control. After culturing with TNF-α for 24 h, cells in six-well plates were harvested to determine the RNA expression of uc.261 by PCR.

Transepithelial electrical resistance assays (TEER) values of Caco2 and T84 cells in Transwell plates were determined with Millicell ERS-2 (Millipore, Billerica, MA, USA), in units of Ω·cm². For more details of TEER assays, see Supplemental Digital Content 1.