Automated fluorescent analysis for cytotoxicity assays

Classical measurements of cytotoxicity using dye exclusion and microscopic evaluation are both time-consuming and inaccurate. Using a cell sorter (TPS) a single dye system has been developed which stains live and complement killed cells with different fluorescence intensity. After exposure of target cells to antibody and complement, ethidium bromide is added to the target cells at a high enough concentration to stain complement killed cells very intensely. The cells are then diluted and lysed to produce single nuclei, permitting live cells to be stained but less intensely than the dead cells. Fluorescence intensity is measured on single nuclei at a rate of 10,000 per minute. For these studies anti-T antisera was titrated for complement dependent cytotoxic activity using normal mouse spleen cells, spleen cells from an anti-thymocyte serum treated mouse, and nylon wool purified mouse splenic T-cells. This procedure makes possible, reliable and reproducible measurement of cytotoxic activity on 5,000 cells per determination.