Horan P K, Kappler J W
J Immunol Methods. 1977;18(3-4):309-16. doi: 10.1016/0022-1759(77)90184-3.
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.
使用染料排斥法和显微镜评估进行细胞毒性的传统测量既耗时又不准确。利用细胞分选仪（TPS）开发了一种单一染料系统，该系统用不同的荧光强度对活细胞和补体杀伤细胞进行染色。在将靶细胞暴露于抗体和补体后，以足够高的浓度向靶细胞中加入溴化乙锭，以使补体杀伤细胞被强烈染色。然后将细胞稀释并裂解以产生单核，使活细胞能够被染色，但染色强度低于死细胞。以每分钟10000个的速率对单核的荧光强度进行测量。对于这些研究，使用正常小鼠脾细胞、抗胸腺细胞血清处理的小鼠的脾细胞以及尼龙毛纯化的小鼠脾T细胞，滴定抗-T抗血清的补体依赖性细胞毒性活性。此程序使得每次测定对5000个细胞的细胞毒性活性进行可靠且可重复的测量成为可能。
