Multspec 1501

Fibroblast-like cells (L929; CRL-6364; ATCC, Manassas, Va., USA) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented by 10% fetal bovine serum, penicillin and streptomycin under standard cell-culture conditions (37 ºC, 100% humidity, 95% air, 5% CO 2 ). The cells were seeded at the desired density (10 4 cells/well) in 96-well plates and incubated for 24 h under standard cellculture conditions to achieve cell attachment before adding the extracts. The cultures then underwent serial extract dilution (undiluted, ½ and ¼). L929 fibroblasts cultured in DMEM without any extracts were used as a control. At 6, 24 and 48 h, the cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. At each time point, an MTT solution (Sigma-Aldrich, St. Louis, MO, USA) was added to the cells and the fibroblasts were incubated at 37 °C for 4 h, shielded from light. The MTT solution was then discarded, and 200 µL of isopropyl alcohol was added to each well. The plate was kept under continuous agitation for 30 min to dissolve the dark blue crystals. The blue solution was then transferred to a 96-well plate to measure the optical density at 570 nm using a spectrophotometer (Shimadzu MultSpec-1501; Shimadzu Corporation, Chiyoda, Tokyo, Japan). Each condition was analyzed in triplicate and according to the ISO 10993-5 (9) .