Ht 29 human colon cells

HT-29 human colon cells (ATCC, USA) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco BRL, USA) containing 10% fetal bovine serum (FBS) (Gibco BRL, USA). Cells were stimulated with or without metformin (5 mM) for 48 h and the lysates were subjected to western blotting. PBMCs from patients with UC were isolated from buffy coats in heparinized blood samples by Ficoll-Hypaque (Amersham Biosciences, UK) density-gradient centrifugation. PBMCs were maintained in RPMI medium containing 10% FBS. PBMCs were stimulated with or without metformin (1 mM) for 72 h and mRNA levels and the number of Th17 cells were examined by real-time PCR and flow cytometry, respectively.

HT29 human colon cells (ATCC, VA, USA) were cultured in RPMI 1640 medium at 37°C/5% CO₂. We treated 2 × 10⁵ HT29 cells with 0.2 or 1 mM metformin (Sigma-Aldrich, St Louis, MO, USA).