MEASUREMENT OF TUMOR-NECROSIS-FACTOR ACTIVITY BY FLOW-CYTOMETRY

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 usi ng flow cytometry. After an incubation with TNF, L929 cells were harve sted and treated with a calcein-AM and ethidium homodimer-1 solution, Nonfluorescent calcein-AM is hydrolyzed by intracellular esterases to yield fluorescent calcein. The ethidium homodimer-1 is a high-affinity red fluorescent DNA dye that is internalized only through altered cel l membranes. A very good correlation was observed between the calcein fluorescence in-tensity and the number of viable cells as well as the ethidium fluorescence and the number of cells with altered membranes. The assay is sensitive, inexpensive, and correlates with the already r eported crystal violet assay while measuring membrane alteration by TN F, It allows the simultaneous measurement of total living and dead cel ls. There is no interference with culture medium components, This meth od is rapid and may be used for routine measurement of TNF activity. ( C) 1995 Wiley-Liss, Inc.