Open AccessMeasurement of tumor necrosis factor activity by flow cytometry
Author(s) -
Lévesque Ann,
Paquet Alain,
Pagé Michel
Publication year - 1995
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990200211
Subject(s) - calcein , flow cytometry , tumor necrosis factor alpha , microbiology and biotechnology , fluorescence , chemistry , propidium iodide , biology , membrane , biochemistry , apoptosis , immunology , programmed cell death , physics , quantum mechanics
Abstract 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.