Morphometric Analysis of Cytolysis in Cultured Cell Monolayers: A Simple and Versatile Method for the Evaluation of the Cytotoxic Activity and the Fate of LAK Cells

Abstract

In vitro techniques for the evaluation of the cytotoxicity of immune cells are important both for the routine assessment of the cytolytic activity in samples for clinical or experimental use and for basic studies of the interaction between killer and target cells. Especially in the latter case, it is important not only to quantify target cell death as an endpoint, but also to observe the interaction and to recover effectors and targets for further analysis. We present a new method that offers considerable improvements for both types of applications, in comparison with the standard radioactivity release assays used today. The morphometric cytotoxicity assay (Mo.C.A.) estimates the extent of target cell lysis by measuring the openings that appear in a confluent monolayer of adherent cells as killed cells detach from the plastic on which they were spread. Two hours after the inoculation of the effector cells, nonadherent killer and dead target cells are washed off and the remaining monolayer is fixed and stained with Coomassie blue. Elementary computer-assisted image analysis allows then to calculate the percentage of open space, which is a parameter for the extent of lysis. As the Mo.C.A. is easy, and does not rely on the use of radioactive compounds or sophisticated equipment, we provide evidence that it should be valuable for the routine analysis of cytotoxicity in various cell samples. In addition, the method offers great flexibility in the choice of target cells and allows for continuous microscopic observation of the live cultures. The interaction can also be stopped at any time, and the effector and (unlabeled) target cells can be recovered separately. Therefore, the method should also offer new possibilities for the basic study of killer cell biology.