MTT assay

In brief, cells were seeded in 96-well culture plates (10×10³cells /200 μl per well) and incubated overnight, and then treated for 24 or 48h with various concentrations of DHA or with the diluting vehicle (DMSO). After incubation, freshly prepared 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (20μl; 5 mg/ml; Sigma) was added to each well and incubated for 4 h at 37°C. The culture medium was removed from the wells and substituted with the reduced MTT solublized in 150μl/well DMSO. The prepared plates were subjected to examination with an Enzyme-linked Immunosorbent Detector (Model 550, Bio-Rad, Hercules, CA, USA) with absorbance values of 490 nm to evaluate cell viability.