A photometric and plaque assay for macrophage mediated tumor cell cytotoxicity

Pretreatment of L-929 cells for 2-3 h with 2 micrograms/ml actinomycin D followed by washing rendered them exceedingly sensitive to the direct cytotoxic effects of murine and rabbit macrophages. We exploited the tremendous increase in sensitivity of L-929 cells to effector cell mediated cytotoxicity by demonstrating simple and convenient photometric and plaque assays capable of being completed in 18 h for the quantitation of macrophage mediated tumor cell killing. The plaques demonstrated were generally visible to the unaided eye and were linearly related to the number of effector cells plated indicating that a plaque represented multitarget cytolysis attributed to a single effector cell. Unlike previously described assays, the effector:target ratios demonstrated were extremely low. Using the described techniques, it was estimated that a single macrophage could kill from approximately 10 to 1000 actinomycin D pretreated and washed target cells. In the presence of LPS-activated effector cells, the majority of these targets that stained with eosin Y did so in a cluster pattern, indicating cytotoxicity rather than mere detachment from the monolayer surface.