[Fluorochromasia lymphocytotoxicity assay for detection of natural killer cell activity using fluorescein-activated...

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.