Temporal Response of Intestinal Epithelium to Microbial Challenges

C2BBe1 cells were seeded in 24-well plates at a density of 1 × 10⁵ cells/well in full DMEM culture medium. Once cells reached confluency, they were cultured in serum and antibiotic-free DMEM overnight (12–16 h) followed by co-culture for 3 h with the commensal E. coli K12, the CD-pathobiont AIEC, the pathogen S. typhimurium [all bacteria strains added at MOI 10:1] as recently described (27 (link)), or a pro-inflammatory cytokine cocktail [consisting of recombinant human (rh) TNF-α (20 ng/ml), rh IFN-γ (10 ng/ml), rh IL-1β (10 ng/ml), all purchased from Peprotech], followed by 3× wash in PBS + 1% Pen/Strep, and a further incubation in full culture DMEM media for 2 (T2), 5 (T5), and 13 (T13) h post infection/cytokine treatment. At each time point, cells were washed with PBS and lysates stored at −80°C for RT-qPCR analyses. All conditions were performed in triplicates.