Cell culture, stress and treatments

The human diploid fibroblast strain IMR-90 was obtained from ATCC. Cells were grown in minimum essential medium (MEM) (Gibco, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco) at 37°C in 5% CO₂. After confluence had been reached, the cells were seeded into 6-well culture plates. One day later, cells were treated for 48 h with 1 mM 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) (Sigma, USA) diluted in MEM + 10% FBS. A 48 h incubation with free radical initiator AAPH establishes a model of oxidative stress-induced cellular senescence [12, 13]. Controls cells were incubated in culture medium alone. After AAPH treatment, IMR-90 were washed with cold phosphate buffer saline (PBS) pH 7.4 and incubated with fresh culture medium or culture medium containing resveratrol (resveratrol group) or MEM supplemented with 3% FBS (CR group) for an additional 48 h before harvest.