Comparison of fluorochrome-labeled and ⁵¹Cr-labeled targets for natural killer cytotoxicity assay

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, BCECF 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 × 10³ 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.