IMPROVED FLUORESCENT BIOASSAY FOR THE DETECTION OF TUMOR-NECROSIS-FACTOR ACTIVITY

Tumor necrosis factor alpha (TNF-alpha) is a monokine of 17 kDa produc ed by activated macrophages and various cells involved in the immune s ystem. We propose a new method for the measurement of TNF activity on mouse L929 fibroblast cells. After an incubation with TNF, the cells w ere stained with a solution of ethidium homodimer-1, a high-affinity r ed 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 b y TNF and not the cell detachment. It requires no rinsing before dye a ddition which may cause cell loss; there is no interference with cultu re medium components since the assay is performed in PBS. This method is more rapid and precise for routine measurement of TNF activity.