Spontaneous increases in the fluorescence of 4,5-diaminofluorescein and its analogs: Their impact on the fluorometry of nitric oxide production in endothelial cells

We studied the spontaneous increase in the fluorescence intensity of 4,5-diaminofluorescein. A slow, steady increase in fluorescence continued for at least 125 h, and this increase was accompanied by ca. 2 nm red shift in the peak of emission spectrum. The spontaneous increase also occurred to diaminorhodamine-4M and a fluorinated form of diaminofluorescein, which has been also used for the detection of nitric oxide (NO). We found that several factors (excitation light, pH etc.) did not alter the time course of this increase. Moreover, we found that this spontaneous increase can produce false-positive results when measuring low-rate nitric oxide production in human umbilical vein endothelial cells, and may confound the interpretation of results of NO production. We show that this adverse effect can be avoided by careful grouping of samples during measurement.