Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain

We have developed a simple, sensitive, fluorescence microplate-based assay for tumor necrosis factor (TNF) biological activity. The assay employs SYTOX Green nucleic acid stain to detect TNF-induced cell necrosis in actinomycin D sensitized cultured cell lines. SYTOX Green stain is a cationic unsymmetrical cyanine dye that is excluded from live cells but can readily penetrate cells with compromised cell membranes. Upon binding to cellular nucleic acids, the dye exhibits a large enhancement in fluorescence, which is monitored at fluorescein wavelengths. We detected 2.5 pg/mL and quantitated 25-500 pg/mL recombinant murine (rm) and recombinant human (rh) TNF-alpha, using mouse fibroblast-derived WEHI 164, WEHI 13var, and L929 cell lines. The procedure can also be used to detect agents that modulate TNF activity. We demonstrated complete inhibition of rhTNF-alpha using monoclonal anti-human TNF-alpha antibody and determined that approximately 20 ng/mL antibody was sufficient to neutralize 50% of the biological activity of 250 pg/mL rhTNF-alpha in these cell lines. Reagents are added in a single step, followed by a 6- to 8-h incubation period, during which the cytokine exhibits its effects. There are no wash steps, and the assay is readily amenable to automation and high-throughput screening procedures.