Cell proliferation assay
In sterile 96-well plate, each well was seeded with ∼1x105 cells. The cells are then grown confluent overnight in 100 μl DMEM with 10% FBS. The medium is replenished with DMEM with 2% FBS and added vehicle control or test substance the next day. For test substance in methanol, stock was added to 25 μl of DMEM with 2% FBS or PBS and placed in the laminar flow hood to let the methanol evaporate (about 1-3 hrs). Once the methanol has evaporated, DMEM with 2% FBS was added to final volume of 100 μl of desired test concentration. A blank methanol control was also made to ensure that any cytotoxicity did not result from methanol contamination. Cells are grown for additional 24 hrs. The cell viability was determined by using Promega CellTiter 96© AQueous One Solution Cell Proliferation Assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) System following the manufacture protocol.