Measurement of tumor necrosis factor activity by flow cytometry
Article first published online: 8 MAR 2005
Copyright © 1995 Wiley-Liss, Inc.
Volume 20, Issue 2, pages 181–184, 1 June 1995
How to Cite
Lévesque, A., Paquet, A. and Pagé, M. (1995), Measurement of tumor necrosis factor activity by flow cytometry. Cytometry, 20: 181–184. doi: 10.1002/cyto.990200211
- Issue published online: 23 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 6 DEC 1994
- Manuscript Received: 11 JUL 1994
- ethidium homodimer-1;
Tumor necrosis factor-α (TNF-α) 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-affinity red fluorescent DNA dye that is internalized only through altered cell membranes. A very good correlation was observed between the calcein fluorescence intensity 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 reported crystal violet assay while measuring membrane alteration by TNF. It allows the simultaneous measurement of total living and dead cells. There is no interference with culture medium components. This method is rapid and may be used for routine measurement of TNF activity. © 1995 Wiley-Liss, Inc.