2.4. Cell culture

Chondrogenic cell line ATDC5 (ATCC, Manassas, VA) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) and plated with a density of 1 ​× ​10⁶ ​cells/mL, supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD). The chondrogenic differentiation medium was supplemented with insulin-transferring-sodium selenite media supplement (Sigma) for 4 days. The cultures were exposed to 25 ​mM (normal) or 50 ​mM (high glucose) d-glucose (Sigma, MO, USA), while 50 ​mM mannitol was used as osmotic control in order to determine whether or not the effect of high glucose was related to the osmotic effect. To address the possible role of NF-ĸB signals, the ATDC5 cells cultured with either 25 or 50 ​mM d-glucose were treated with 5 ​μM Bay-117082 (NF-ĸB inhibitor, Thermo Fisher Scientific, USA).