Morphometric analysis of cytolysis in cultured cell monolayers: a simple and versatile method for the evaluation of the lytic activity and the fate of LAK cells

Abstract

The assessment of cytolytic activity of killer cells may not only be useful to improve routine analysis, e.g., in clinical settings, but may also offer new opportunities for the fundamental analysis of the mutual interaction between cytotoxic cells and their targets. We have developed a morphometric method to estimate cytolytic activity of activated natural killer (NK) cells by measuring the clearance of a precultured confluent monolayer of adherent target cells, e.g., immortalized fibroblasts. Cytotoxic cells are inoculated on top of confluent monolayers of target cells and after 2 h, nonadherent cells are washed off and intact adherent cells are fixed and stained with a Coomassie blue solution. Elementary computer-assisted analysis of the resulting microscopic images and measurement of the cleared area provide us with a sensitive and reproducible parameter of target cell lysis. We found that the assay can be used with targets of very different origin, as long as they form confluent monolayers, and with different populations of killer cells. The morphometric cytotoxicity assay (MoCA) offers several advantages: storage of samples for postponed analysis, increased sensitivity as compared to radioactive assays, continuous visualization during assay, availability of targets and effectors for subsequent analysis after interaction.