Colorimetric cell viability assay (MTT). Solubilization step is required.

• A Colorimetric Assay to Assess Cell Viability
• Greater sensitivity than the neutral red procedure
• Non-radioactive, fast and convenient assay applicable to a variety of cells

The CellTiter 96 Assay is a collection of qualified reagents that provide a convenient method of determining viable cell number. The CellTiter 96 Assay is a modification of the MTT assay method described by Mosmann and incorporates several improvements to the method that address previous technical problems including: 1) serum protein precipitation caused by adding organic solvent; 2) interference by phenol red; 3) incomplete solubilization of the formazan crystals resulting in lower sensitivity; and 4) stability of the colored product. The CellTiter 96 Assay is performed by adding a premixed, optimized Dye Solution to culture wells of a 96-well plate, usually containing various concentrations of growth factor or test substance. During a 4-hour incubation, living cells convert the MTT tetrazolium component of the Dye Solution into a formazan product. If you currently use a [(3)H]-thymidine incorporation assay, the addition of Dye Solution can be substituted for the pulse of radioactive thymidine at the time point in the assay when the pulse of [(3)H]-thymidine is usually added. The Solubilization/Stop Solution is then added to the culture wells to solubilize the formazan product, and the absorbance at 570nm is recorded using a 96-well plate reader. In addition, direct comparison between [(3)H]-thymidine incorporation and tetrazolium conversion have demonstrated less than a 5% difference between the two assays for determination of growth factor content of several samples.

During a four-hour incubation, living cells convert the MTT tetrazolium component of the Dye Solution into a formazan product. If you currently use a [3H]-thymidine incorporation assay, the addition of Dye Solution can be substituted for the pulse of radioactive thymidine at the time point in the assay when the pulse of [3H]-thymidine is usually added. The Solubilization/Stop Solution is then added to the culture wells to solubilize the formazan product, and the absorbance at 570 nm is recorded using a 96-well plate reader. In addition, direct comparison between [3H]-thymidine incorporation and tetrazolium conversion have demonstrated less than a 5% difference between the two assays for determination of growth factor content of several samples.

The CellTiter 96 Assay is performed by adding a premixed, optimized Dye Solution to culture wells of a 96-well plate that usually contains various concentrations of growth factor or test substance.