A flow cytometric long-term cytotoxicity assay.

A method to evaluate cytotoxic effects applicable over a wide range of incubation times has been developed. It is based on quantification by flow cytometry of dead and viable target cells stained by covalently binding the fluorescent dye fluorescein isothiocyanate (FITC). The staining with FITC did not affect cell viability and growth parameters and was stable enough to identify target cells for at least 2 days. The flow cytometric analysis of the cell mixture was performed in a test system with activated CD8+ lymphocytes as effector cells and melanoma M21 cells as targets in the presence of appropriate bispecific antibodies and revealed a rather complex pattern composed of several distinct cell subsets which were identified by use of antibodies to lymphocyte antigens. The assay compared favourably with results from a conventional 51Cr release assay obtained after 4 h and 8 h of incubation and from a target cell adherence assay obtained after 24 h of incubation. The application of the method described herein is especially advantageous for the evaluation of long-term cytotoxic effects. Furthermore, it provides valuable multi-parameter information which is useful for elucidating mechanisms of cytotoxicity.