In order to determine the cytotoxicity of different anti-viral agents in our cell culture, we have been using the Promega CellTiter 96 Non-Radioactive Cell Proliferation Assay. This assay provides us with a quick, simple way to determine the number of viable cells following treatment with different drugs at a variety of time points. This kit uses the cellular conversion of tetrazolium salt into a formazan product, which can then be detected using our 96-well plate reader.
The kit simply consists of two reagents: The Dye Solution, containing tetrazolium salt, and the Stop Solution. Frequent freezing and thawing should be avoided, so we aliquot into microtubes and store them at –20 C. Also, the Dye Solution is light-sensitive, so exposure to light should be minimized. The Stop Solution can be left on your bench at room temperature and is stable for approximately 6 months.
The kit is very easy to use. Basically, we aliquot the appropriate amount of cells (in our case 50,000 cells/well) into 96-well plates. We treat them with our drugs for a fixed amount of time. The pre-mixed Dye Solution is then added to determine the cytotoxicity of treated to untreated cells. The 96-well plate is incubated in the 37 C tissue culture incubator for 3-4 hours, allowing the living cells to convert tetrazolium (in the dye) to a formazan product. The Stop Solution is added to each sample and mixed well with a pipettor in order to solubilize the formazan product. After incubation with the Stop Solution at room temperature for 1 hour, the absorbance is read at 590 nm using our 96-well plate reader. Conveniently, incubation with the Stop Solution can be extended as long as overnight if needed without appearing to significantly affect absorbance compared to 1 hour incubation.
The advantage of this kit compared to radioactive incorporation assays is that it does away with the use of radioactive isotopes, eliminating extra cost, paper work and disposal concerns associated with radioactivity. The advantage of this kit over conventional cell counting (i.e. manual counting of viable cells using a microscope) is that we can use a 96-well format and reader, allowing us to obtain computerized data quickly. In addition, the amount of work is reduced because this protocol does not involve tedious steps requiring washing and removal of solution from wells. Importantly, the elimination of these washing steps also reduces the possibility of cell loss during the washing steps. This assay is applicable for both anchorage-dependent and suspension cells hence, this kit is very convenient.
The cost of this kit is expensive (a little over $200), but considering the number of assays that can be done (1000 assays) it is well worth its price. I recommend without reservation this Promega assay kit for determining cell cytotoxicity due to its efficiency and simplicity.
Hee Chul Lee, Ph.D.
Post-Doctoral Scientist
Dept. of Biochemistry
NYU School of Medicine