Wierda W G, Mehr D S, Kim Y B
Department of Microbiology and Immunology, University of Health Sciences, Chicago Medical School, IL 60064.
J Immunol Methods. 1989 Aug 15;122(1):15-24. doi: 10.1016/0022-1759(89)90329-3.
An alternative method for measuring in vitro cellular cytotoxicity has been developed utilizing the carboxyfluorescein derivative 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) as the target cell label. Target cells labeled with the fluorescent dye are incubated with effector cells, if killing of targets occurs, BCEDF is released analogous to 51Cr release. Measurement of specific lysis in this assay is based on the direct measurement of dye retained by the remaining viable target cells using the Pandex FCA. In paired experiments we have compared the fluorochrome assay to the standard 51Cr release assay in measuring porcine natural killer cytotoxicity. The target labeling time with BCECF is 30 min as opposed to 1 h with 51Cr; and there is no significant dye reincorporation after release. The optimal target number per incubation well for the BCECF assay is 5 X 10(3) cells. In both the BCECF and 51Cr release assays, maximum percent specific lysis is reached after 3-4 h incubation. By 2 h incubation, the BCECF assay reaches the maximum seen with 51Cr and in a 4 h assay the maximum NK activity measured with BCECF labeled targets is always higher than that measured with 51Cr-labeled targets. In paired experiments, we have shown the reproducibility of the BCECF assay and that the BCECF assay measures NK enhancement by NK enhancing monoclonal antibody and inhibition by NK inhibiting monoclonal antibody as good as the 51Cr release assay, if not better. In conclusion, the BCECF assay is a reliable and reproducible measure of in vitro cellular cytotoxicity, eliminates the use of radioisotopes and is cost efficient.
已开发出一种用于测量体外细胞毒性的替代方法，该方法利用羧基荧光素衍生物2',7'-双（羧乙基）-5,6-羧基荧光素（BCECF）作为靶细胞标记物。用荧光染料标记的靶细胞与效应细胞一起孵育，如果发生靶细胞杀伤，BCECF会像51Cr释放一样被释放出来。该检测中特异性裂解的测量基于使用Pandex FCA直接测量剩余存活靶细胞保留的染料。在配对实验中，我们将荧光染料检测与标准的51Cr释放检测在测量猪自然杀伤细胞毒性方面进行了比较。用BCECF标记靶细胞的时间为30分钟，而用51Cr标记则为1小时；释放后没有明显的染料再掺入。BCECF检测每个孵育孔的最佳靶细胞数量为5×10(3)个细胞。在BCECF和51Cr释放检测中，孵育3 - 4小时后达到最大特异性裂解百分比。孵育2小时时，BCECF检测达到了用51Cr检测时所见的最大值，并且在4小时的检测中，用BCECF标记靶细胞测量的最大自然杀伤细胞活性总是高于用51Cr标记靶细胞测量的活性。在配对实验中，我们已证明BCECF检测具有可重复性，并且BCECF检测在测量自然杀伤细胞增强单克隆抗体增强自然杀伤细胞活性以及自然杀伤细胞抑制单克隆抗体抑制活性方面，即便不比51Cr释放检测更好，也与它一样好。总之，BCECF检测是一种可靠且可重复的体外细胞毒性测量方法，无需使用放射性同位素且成本效益高。
