Enhancing Formazan Dissolution to Improve Accuracy in MTT‑Based Cell Viability Assays

The 3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a method that used to measure cell viability. It is based on the conversion of MTT by metabolically active cells succinic dehydrogenase enzyme into water insoluble formazan. Dissolution of formazan by using proper solvent is the most important step of MTT assay to obtain valid and reliable data. In this study, various solvents were assessed to facilitate faster, less labor-intensive, and more reliable dissolution of formazan. The solvents tested included dimethyl sulfoxide (DMSO), DMSO supplemented with 0.005 N hydrochloric acid (HCl), 10% sodium dodecyl sulfate (SDS) in water containing 0.005 N HCl, 5% SDS with 0.005 N HCl in DMSO, and 10% SDS with 0.005 N HCl in DMSO. Formazan solubility was assessed using BALB/3T3 cells. Following the addition of MTT, cells were incubated for 4 h at 37˚C, after which formazan crystals were dissolved either with or without medium, and absorbance was measured at 570 nm. Among the solvents compared, DMSO was the most effective for formazan dissolution in the absence of medium, whereas 10% SDS with 0.005 N HCl in DMSO yielded the highest optical density (OD) in the presence of medium. The lowest solubility was observed with 10% SDS and 0.005 N HCl in water. Additionally, the improved MTT assay eliminates the requirement for medium removal, hence enabling its employment under both conditions—with or without medium. This experimental improvement simplifies the adaptability of the MTT assay for adherent cell lines and streamlines its application in suspension cell lines, eventually improving its overall utility while simplifying the experimental procedure.