Colon were collected from 6 to 10 weeks Ahr+/+ and Ahr−/− mice. Organs were cleaned of feces by flushing with PBS 2% Fetal bovine serum (FBS) using a syringe and were cut first longitudinally and then laterally into 0.5 cm length pieces. Each tissue pieces were incubated for 30 min at 37◦C in 10 mL digestion solution containing DMEM with 5% FBS, 10 mM HEPES (Euroclone S.p.a Milan, IT), 5 mM EDTA, 1 mM DTT (Sigma-Aldrich, St. Louis, MO, USA). After incubation, samples were mixed for 30 s using a vortex mixer. Cell suspensions were stored on ice and supernatant were separate from colon tissue pieces and transferred into new tube after were applied into a MACS SmartStrainer 100 µm (Miltenyi Biotec, Bergisch Gladbach, GE) placed on a 15 mL tube. After centrifugation cell suspension were applied onto a MACS SmartStrainer 75 µm (Miltenyi Biotec, Bergisch Gladbach, GE) for remove impurity and resuspended in DMEM containing 10% FBS, 1% l-glutamine, and 1% penicillin/streptomycin.

Approximately 1 × 106 extracted cells were seeded in 24 well plate and incubated at 37 °C for 2 h. After incubation IEC cells were treated with mTNF-α (Sino Biological, Beijing, CN) 100 ng/mL for 20 h alone or in combination with Pelargonidin (5, 10, and 20 µM) and mNF-κB inhibitor (Sigma-Aldrich, St. Louis, MO, USA) 100 nM for gene expression profile analysis.

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