Aida H, Suzuki S, Ikeda T, Kanayama M, Yamanaka M
Central Laboratory, Yokoisuka Kyosai Hospital.
Rinsho Byori. 1990 Mar;38(3):301-5.
A new fluorochromasia lymphocytotoxicity assay using a fluorescein-activated cell sorter (FACS) was developed for detecting natural killer cell (NK) activity. Carboxy-fluorescein-diacetate (C-FDA) labeled K 562 cell was used as the target cell. An optimal labeling condition is incubation with 25 micrograms/ml of C-FDA for 1 hour to separate target cells from effector cells by FACS. These labeled cells were cocultured with peripheral blood mononuclear cells (PBMC) as effector cells or with heat-activated PBMC as control cells. At various effector/target cell ratios, the number of C-FDA positive cells determined by FACS showed good reproducibility (coefficient of variation ranged from 0.9 to 9.1%), and an incubation period of 4 hours was sufficient for lysis. At the end of the lysis period, the number of target cells cultured with effector cells (A) or with control cells (B) was determined by FACS. Percent NK activity was calculated according to the following formula: (1-A/B) x 100. An adequate correlation between NK activity assayed by the 51Cr-method (x) and C-FDA (y) on the same cell population simultaneously was obtained (r = 0.89, the regression curve was y = 0.86 x + 3.74). C-FDA assay using FACS appears to be a good alternative to the 51Cr assay in the detection of NK activity.
为检测自然杀伤细胞（NK）活性，开发了一种使用荧光激活细胞分选仪（FACS）的新型荧光染色淋巴细胞毒性测定法。用羧基荧光素二乙酸酯（C-FDA）标记的K 562细胞作为靶细胞。最佳标记条件是与25微克/毫升的C-FDA孵育1小时，然后通过FACS将靶细胞与效应细胞分离。将这些标记细胞与作为效应细胞的外周血单个核细胞（PBMC）或与热激活的PBMC作为对照细胞共培养。在不同的效应细胞/靶细胞比例下，通过FACS测定的C-FDA阳性细胞数量显示出良好的重复性（变异系数范围为0.9%至9.1%），4小时的孵育期足以实现裂解。在裂解期结束时，通过FACS测定与效应细胞（A）或对照细胞（B）共培养的靶细胞数量。NK活性百分比根据以下公式计算：(1 - A/B)×100。在同一细胞群体上同时通过51Cr法（x）和C-FDA（y）测定的NK活性之间获得了充分的相关性（r = 0.89，回归曲线为y = 0.86x + 3.74）。在检测NK活性方面，使用FACS的C-FDA测定法似乎是51Cr测定法的一个很好的替代方法。
