The CellTiter 96® AQueous One Solution Cell Proliferation Assay is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays. The CellTiter 96® AQueous One Solution Reagent contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine ethosulfate; PES). PES has enhanced chemical stability, which allows it to be combined with MTS to form a stable solution. The assay uses phenazine methosulfate (PMS) as the electron coupling reagent, and PMS Solution and MTS Solution are supplied separately.
Assays are performed by adding a small amount of the CellTiter 96® AQueous One Solution Reagent directly to culture wells, incubating for 1–4 hours and then recording absorbance at 490nm with a 96-well plate reader. The quantity of formazan product as measured by the amount of 490nm absorbance is directly proportional to the number of living cells in culture.
If you currently use a [3H]-thymidine incorporation assay, addition of the CellTiter 96® AQueous One Solution Reagent can be substituted for the pulse of [3H]-thymidine at the time point in the assay when the pulse of radioactive thymidine is usually added. Previous bioassay data comparing [3H]-thymidine incorporation to the MTS-based CellTiter 96® AQueous Assay and the original MTT-based CellTiter 96® Assay demonstrate that tetrazolium reagents can be substituted for [3H]-thymidine incorporation.
Features and benefits
The simple "add-incubate-measure" format (single-step reagent addition) enables design of homogeneous high-throughput screening assays. Unlike MTT, the assay can be used as a single solution and is filter sterilized and ready to add to assay plates. Fewer steps are needed as the assay can be performed in 96-well plates with no washing or cell harvesting steps. Additionally, solubilization steps normally required for MTT assays are eliminated.
The assay provides flexibility as plates can be read and returned to an incubator for further color development, and with the elimination of volatile organic solvent to solubilize the formazan product and radioactive reagents commonly required of similar assays, the CellTiter 96® AQueous One Solution Cell Proliferation Assay is safer to use.