A new fluorometric assay for cytotoxicity measurements in-vitro.

Cytotoxicity testing of anticancer drugs requires techniques which are sensitive, reproducible and applicable to large scale testing using automated instruments. These assays are presently performed with end point staining of cell proteins with dyes, viability stains or energy dependent of substrates such as MTT or XTT. Although reliable. these assays are not sensitive enough, too expensive for large scale screening or they use reagents that may be harmful for personnel or equipment. We describe, the use of Alamar Blue, a new non fluorescent substrate, which after reduction in living cells, yields a very strong fluorescent product. Using the automated fluorescence plate reader Cytofluor, we have evaluated the various parameters such as substrate concentration, time and volume of incubation with respect to linearity and lower limit of detection. We found that for a two hour assay, this new non toxic substrate could detect as low as 200 cells per well with a useful measurement range up to 20,000 cells per well. The fluorescent assay is more than ten times as sensitive as the colorimetric assay. When the cytotoxicity of daunorubicin was measured with this assay and compared to the XTT formazan assay we found comparable IC50 values but this new assay was more economical and results are obtained in two hours as compared to four hours for the formazan assay. This new economical and versatile assay could be used with advantage for large scale in vitro screening of anticancer drugs and other cytotoxic agents.