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 SYTO X Green nucleic acid stain to detect TNF-induced cell necrosis in actinomyc in D sensitized cultured cell lines. SYTOX Green stain is a cationic unsymm etrical cyanine dye that is excluded from live cells but can readily penetr ate cells with compromised cell membranes. Upon binding to cellular nucleic acids, the dye exhibits a large enhancement in fluorescence, which is moni tored at fluorescein wavelengths. We detected 2.5 pg/mL and quantitated 25- 500 pg/mL recombinant murine (rm) and recombinant human (rh) TNF-alpha, usi ng 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 similar to 20 ng/mL antibody was su fficient to neutralize 50% of the biological activity of 250 pg/mL rhTNF-al pha 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 effect s. There are no wash steps, and the assay is readily amenable to automation and high-throughput screening procedures. (C) 2001 Academic Press.