MTT assay for cytotoxicity and cell viability determination

The tetrazolium MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Acros Organics, Belgium) was dissolved in phosphate buffered saline (PBS, Sigma-Aldrich, St. Louis, MO) at a concentration of 5mg/mL. The MTT solution was then filter-sterilized using a 0.22micron syringe filter (Merck Millipore, Billerica, MA) and placed in a light-protected bottle. To each well of a 96-well plate containing human 3A placenta cells, 20μL of the MTT solution was added and the cells were incubated at 37°C for 3.5 hours. The media was removed and 150μL MTT solvent [4mM hydrochloric acid (Sigma-Aldrich, St. Louis, MO), 0.1% NP-40 substitute (US Biological, Salem, MA) in isopropanol (EMD Millipore, Billerica, MA)] was added. The plate was then covered with aluminum foil and incubated on a shaker at room temperature for 15 minutes. Absorbance at 590nm was measured in triplicate using a Tecan GenIOS Pro plate reader (Tecan, Durham, NC)