Automated fluorescent analysis for drug-induced cytotoxicity assays

The human tumor clonogenic assay has been reported to predict for sensitivity of human tumors to a variety of drugs. However, this assay requires large numbers of viable cells, is time-consuming, and takes at least 2 weeks before results are available. To circumvent these problems, Weisenthal developed a microscope-based dye exclusion assay. Because this method is also time-consuming and subject to observer error, we have developed an automated method of quantitating drug cytotoxicity using a flow cytometric cell sorter (FCM). After incubation of drug-exposed tumor cells, acetaldehyde-fixed duck red blood cells (DRBC) are added. Dead tumor cells and the fixed DRBC are stained by the fluorescent dye propidium iodide, which penetrates dead cell membranes. A two-parameter analysis (cell size as measured by narrow angle light scatter vs propidium iodide fluorescence) enables determination of the live tumor cell:DRBC ratio. There was a strong correlation between the FCM method and manual counting (r = 0.958 for cell lines, r = 0.831 for fresh leukemic cells, P < 0.0001 in both cases). We conclude that the automatized FCM method gives compatible results to the manual dye exclusion assay and increases efficiency.