A rapid extremely sensitive, quantitative microassay for cytotoxic cytokines

Pretreatment of the murine mammary adenocarcinoma cell line, EMT-6, with low levels (0.8-1.2 micrograms/ml) of actinomycin D, prior to incubation with a spectrum of cytotoxic cytokines, converted this target from marked resistance to extreme sensitivity. The drug-treated EMT-6 was from 5-50 fold more sensitive to cytokine attack than the widely used actinomycin D-treated murine L-929 target. Drug-induced growth inhibition allowed evaluation solely of the cytolytic effects of these cytokines. Lysis was evaluated after a 16-24 hr incubation by the uptake by viable cells of neutral red or by their reduction of [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. This microcytotoxicity assay should facilitate purification and characterization of cytotoxic cytokines, the purification of their mRNAs in translation systems and the detection of the expression of their encoding genes.