ABSTRACT
Introduction: There are conflicting data on the ability of human mononuclear cells to produce nitric oxide (NO). We investigated nitric oxide production from peripheral blood mononuclear cells (PBMs) by using a new sensitive fluorescent indicator.
Materials and Methods: PBMs from healthy volunteers were collected, plated in 96-well microplates, and loaded with the fluorescent nitric oxide probe, 4,5-diaminofluorescein diacetate (DAF-2DA). Experiments were performed in normal control and endotoxin-stimulated PBMs, with and without exogenous L-arginine. The exogenous nitric oxide donor S-nitroso-N-acetyl-penicillamine (SNAP) was used as a positive control. Fluorescence intensity was measured with a fluorescence microplate reader. Results: Nitric oxide production by human PBMs can be demonstrated by the use of the fluorescent indicator, DAF-2DA, in both control and endotoxin-stimulated conditions. Nitric oxide production was independent of the concentration of exogenous L-arginine. The addition of endotoxin did not change nitric oxide production. PBMs treated with SNAP showed a concentration dependent increase in fluorescence. Nitric oxide production over 5 hours was constant and identical in both control and stimulated groups. Conclusion: This fluorescent indicator technique is useful for the study of NO production by human PBMs. Nitric oxide production by PBMs was independent of exogenous L-arginine concentration and was not affected by endotoxin.The discovery of nitric oxide as a biomolecule with important physiological function has led to the development of various analytical methods for its detection and quantification. To measure nitric oxide generated under physiological conditions several different fluorimetric methods which were originally developed for nitrite (NO<sub>2</sub><sup>-</sup>) and nitrate (NO<sub>3</sub><sup>-</sup>) determination have been employed.
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