Related Experiment Videos
Nitric oxide production by human peripheral blood mononuclear cells
1Department of Anesthesiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700 Thailand. siptm@mahidol.ac.th
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.