MEASUREMENT OF CYTOTOXICITY BY PROPIDIUM IODIDE STAINING OF TARGET-CELL DNA - APPLICATION TO THE QUANTIFICATION OF MURINE TNF-ALPHA

A rapid and sensitive method is described for the determination of mur ine tumor necrosis factor (TNF-alpha), which can be performed in micro titer plates using a fluorescence plate scanner. The method is based o n the binding of propidium iodide (PI), a membrane-impermeant dye, to nucleic acids of WEHI 164 cells, whose plasma membrane becomes permeab le due to TNF-alpha-induced cell damage. The analytical range for the proposed method is 0.3-200 pg/ml of TNF-alpha after 5 h of incubation. The optimal number of target cells was found to be 4-5 X 10(4)/well. The variability obtained for the PI assay was 7.6%; lower than that ob tained with a commonly employed method in which MTT is used to determi ne cell viability (11.3%), Thus, the PI assay appears to be a reliable and reproducible method for the determination of biologically active TNF-alpha. The assay can be performed in a few hours and has the advan tage over the current MIT and Cr-51-release assays that kinetic studie s of TNF-alpha toxicity are possible since it permits multiple, sequen ced measurements of cell viability during the incubation of the sample . The method can also be used for the determination of human TNF-alpha .