The Promega CellTiter 96® AQueous One Solution Cell Proliferation Assay uses a colorimetric method to determine the number of viable cells in proliferation or cytotoxicity assays. This method can be used to replace the classic 3
H-thymidine incorporation assay. The one solution assay system contains a novel tetrazolium compound, MTS, and an electron coupling reagent, PES. PES combines with MTS and confers enhanced chemical stability and allows the formation of a stable solution.
The principle of the assay is that the MTS tetrazolium compound is reduced in cells into a colored formazan compound which is soluble in tissue culture media. This conversion occurs via dehydrogenase enzymes which are present in live cells. The assay is straight-forward and simple. Essentially, cells are plated in a 96-well cell culture plate and allowed to grow over a pre-determined period of time, typically 48-72 hrs, in 100 ul of culture medium. At the end of the incubation period, the CellTiter 96® AQueous One Solution is thawed and 20 ul is added to each well. The plate is incubated for a further 1-4 hrs at 37ºC in a humidified CO2 incubator. The absorbance is then read at 490 nm with a microplate spectrophotometer. Repeat readings of the absorbance at 490 nm can be taken during the 1-4 hr time course. Alternatively, the reaction can be stopped by the addition of 25 ul of 10% SDS and read up to 18 hrs later. The quantity of formazan produced when measured by absorbance at 490 nm is directly proportional to the number of viable cells in culture. Unlike other proliferation assays employing a formazan product such as MTT or INT which form a crystalline precipitate, the MTS formazan product is soluble in tissue culture medium thus obviating the need for extra steps to dissolve precipitates prior to reading absorbance.
Typically, we have used this reagent to determine the toxicity or anti-proliferative characteristics of a chemical entity. It should be noted that the reagent would also function well to determine the effect of a growth factor which increases growth. It is possible to determine the IC50or ED50 of such an entity by plotting the concentration of the chemical entity against the absorbance at 490 nm. We have found that the AQueous One Solution reagent works with a wide range of cell types (We have used it with COLO205, A549, HeLa, MCF-7) and different media (RPMI, DMEM, MEM, F12).
Prior to using this reagent, it is necessary to determine the optimal cell number to plate at the start of the experiment. Due to the fact that cells grow during the course of the experiment (48-72 hrs); it is necessary to use an initial cell number that will produce a absorbance reading which is within the linear range of the assay. The linear range of the assay can be determined by culturing various starting cell numbers and measuring the absorbance at 490 nm after the incubation period and choosing a cell number which gives a absorbance reading at the lower end of the linear range. Promega suggest that 5000 cells/well works well for most cell types and this has been our experience.
In summary, the CellTiter 96® AQueous One Solution is a very easy-to-use and simple solution to determine cell proliferation. The fact that radioactivity is not used is an added advantage, as is the fact that organic solvents are not necessary to solubilize the formazan product, all of which adds to the convenience of this reagent.