Abstract

AIM. Analysis of cytotoxic functions is required for characterization of cells for cell-based immunotherapy. However, the radioisotope-based methods used so far require specific working conditions and equipment. In order to optimize a nonradioactive method for analysis cytotoxic function of in vitro cultured natural killer (NK) cells for use in clinical laboratories, we investigated use of cytometry.

METHODS. By using CFSE and 7-AAD double labelling of target cells and simultaneous multiparameter analysis of effector cells, we optimized the analysis of cytotoxicity as a cytometry-based assay.

RESULTS. Our results established the useful range of effector/target ratio to be 0.1 to

10. We found that cytolysis depends on the effector/target ratio according to the law of mass action. The in vitro cultivated NK cells are highly cytotoxic, yet their function is regulated by target cell histocompatibility antigens (MHC I). The standard error of our assays at Cmax was 2.0–2.6% and 95% confidence interval ±<5%.

CONCLUSION. We conclude that cytometric analysis of cytotoxic function is highly sensitive, consistent, and well suited for laboratories not equipped for work with radioisotopes.