Evaluation of monensin and brefeldin A for flow cytometric determination of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha in monocytes

Flow cytometry has become a powerful technique to measure intracellular cyt okine production in lymphocytes and monocytes, Appropriate inhibition of th e secretion of the produced cytokines is required for studying intracellula r cytokine expression. The aim of this study was to compare the capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (interleukin-1 beta [IL-1 beta], IL-6, tumor necrosis f actor-alpha [TNF-alpha]) within peripheral blood monocytes. A two-color flo w cytometric technique was used to measure intracellular spontaneous and li popolysaccharide (LPS)-stimulated IL-1 beta, IL-6, and TNF-alpha production in monocytes (CD14+) of whole blood cultures. The viability of monensin-tr eated monocytes was slightly lower than that of brefeldin A-inhibited monoc ytes, as measured with propidium iodide (PI), The percentage of IL-6 and TN F-alpha -producing monocytes after 8 h of culture without stimulation revea led significant lower values for monensin-treated than for brefeldin A-trea ted monocytes, The percentages for stimulated cells did not differ. The spo ntaneous intracellular production in molecules of equivalent soluble fluoro chrome units (MESF) of IL-1 beta, IL-6, and TNF-alpha after 8 h of culture was higher in brefeldin A than in monensin-inhibited monocytes, The LPS-sti mulated intracellular production of IL-1 beta, IL-6, and TNF-alpha was incr eased in brefeldin A-inhibited monocytes, In conclusion, for flow cytometri c determination of intracellular monocytic cytokines (IL-1 beta, IL-6, and TNF-alpha), brefeldin A is a more potent, effective, and less toxic inhibit or of cytokine secretion than monensin. (C) 2001 Wiley-Liss, Inc.