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.