Improved fluorescent bioassay for the detection of tumor necrosis factor activity

Tumor necrosis factor alpha (TNF-alpha) is a monokine of 17 kDa produced by activated macrophages and various cells involved in the immune system. We propose a new method for the measurement of TNF activity on mouse L929 fibroblast cells. After an incubation with TNF, the cells were stained with a solution of ethidium homodimer-1, a high-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. The assay is sensitive, inexpensive and correlates with the already reported TNF assays while measuring the membrane alteration by TNF and not the cell detachment. It requires no rinsing before dye addition which may cause cell loss; there is no interference with culture medium components since the assay is performed in PBS. This method is more rapid and precise for routine measurement of TNF activity.