Bottom chamber

Caco-2 were purchased from ATCC (ATCC, USA). HT29-MTX E12 cells were obtained through Millipore/Sigma (Merck, USA) via the European Collection of Authenticated Cell Cultures and maintained in DMEM with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM L-glutamate and 1% MEM amino acids solution (Cytiva, USA). Prior to cytokine exposure, cells were cultured at 8% CO₂, 37 °C until 80% confluent with media changes every other day. HT29-MTX E12 cells were cultured on 0.4 μm pore Transwell® filter systems (Corning, USA). For standard liquid interface cultures (LI), 250 μL growth media was placed in the top chamber and 500 μL in the bottom. LI cultures treated with cytokine after monolayers achieved TER of ~ 300 Ω·cm². ALI conditions were generated by removal of media from the top chamber once the above-mentioned TER was achieved. After transition to ALI, cells were then treated with TNF-α and IFN-γ at 2 ng/ml each for 48 h unless otherwise indicated (bottom chamber, BioVision, USA).