A new colorimetric method for measuring cell-mediated cytotoxicity in dogs

Abstract

In order to replace the radioactive 51chromium release assay (CRA), a colorimetric method for the determination of cell-mediated cytotoxicity of natural killer (NK) effector cells of dogs and adherent target cells was developed using the dye Rose Bengal (RB). After a 14 h incubation period of leucocytes isolated from the peripheral blood (PBL) of dogs and a natural killer cell-sensitive canine adenocarcinoma cell line (CTAC), effector and lysed target cells were removed by washing, and the surviving adherent target cells were stained with RB. The optical density (OD) of the remaining target cells was measured in a microspectrophotometer (ELISA reader) and was found to correspond to the number of surviving cells, and thus was inversely correlated to the cytotoxic activity. The RB assay revealed almost identical cytotoxic values when compared with the CRA. In contrast to this assay the RBA is quick and easy to perform, inexpensive and avoids radioactive materials and waste. However, the method is restricted to adherent target cells.