Tumor necrosis factor-a (TNF-a) 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 using flow cytometry. After an incubation with TNF, L929 cells were harvested 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-af€inity red fluorescent DNA dye that is internalized only through altered cell membranes. A very good correlation was observed between the calcein fluorescence inTumor necrosis factor was originally defined as a factor produced in animals primed with Bacillus CalmetteGuerin (BCG) in response to endotoxin (4). Tumor necrosis factor-a (TNF-a) plays a central role in the host immunological response against various bacteria and parasitic infections (2). It was found to be responsible for the necrosis of various tumors and to be a cytotoxic factor produced by T cells (6). Cells of the mononuclear phagocyte lineages are the primary source of TNF-a, but it is now known that many other cell types produce this cytokine, including lymphocytes, mast cells, and even fibroblasts ( 15). TNF is involved in various pathological and physiological situations, and it is implicated alone or in association with other cytokines such as interleukin (1L)-I , IL-6, and interferons in the pathogenesis of different diseases such as septic shock and cerebral malaria and autoimmune diseases such as rheumatoid arthritis (17,10,7).Various assays have been reported for the measurement of TNF cytotoxicity (1,3,5,8,9,(11)(12)(13)16 Recombinant TNF The recombinant human TNF-a used for these assays was produced in our laboratory (14). This purified TNF-a had a specific activity of 10' units per milligram as determined by the standard T N F assay of Aggarwal and Kohr ( 1 ). One unit is defined by the quantity of TNF that produces 50% lysis of L929 fibroblasts.
Optimization of Dye Concentrations and Incubation TimeMouse L929 fibroblasts were trypsinized and washed three times; 5 X lo4 cells were seeded in a 96-well flatbottom microtiter plate in 100 pl phosphate-buffered saline (PBS), pH 7.2. Various concentrations of either calAddress reprint requests to Michel Pa&