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J Leukoc Biol. 1992 May;51(5):496-500.

Flow cytometric analysis of nitric oxide production in human neutrophils using dichlorofluorescein diacetate in the presence of a calmodulin inhibitor.

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  • 1Geriatric Research, Education and Clinical Center, Veterans Affairs Center, Durham, North Carolina 27705.


Dichlorofluorescein (DCFH) oxidation assay measures hydrogen peroxide (H2O2), which is a derivative of superoxide anion. We found that a calmodulin antagonist, W-13, which is known to inhibit superoxide anion generation enhanced the capacity of human neutrophils to oxidize DCFH. To investigate this discrepancy we studied the role of nitric oxide (NO) in DCFH oxidation. Pure NO was capable of oxidizing DCFH, and the product formed had spectral properties identical to oxidized DCFH produced by H2O2. The arginine analog, NG-monomethyl-L-arginine (NMMA), which inhibits NO production, in combination with W-13 completely inhibited the stimulus-induced increase in DCFH oxidation. We conclude that the oxidation of DCFH in human neutrophils can occur by either H2O2 or NO.

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