Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5‐diaminofluorescein (DAF‐2): implications for cellular NO imaging

Abstract

The fluorescent indicator of nitric oxide (NO), 4,5-diaminofluorescein (DAF-2), and its membrane-permeable derivative (DAF-2 diacetate) have been recently developed to perform real-time biological imaging of NO. In this study, we show that DAF-2 is strongly influenced by factors other than the concentration of NO itself. Using measurements with a fluorimeter as well as fluorescence microscopy, we found that the divalent cation concentration in the medium, as well as the incident light, strongly affects the ability of DAF-2 to detect NO. Calcium, in particular, enhanced the signal detection of NO released by NO donors by up to 200 times. With multiple and longer exposures to light, no bleaching of the dye was observed but, instead, a potentiation of the fluorescence response could be measured. While these two properties will affect the use and interpretation of the hitherto acquired data with this fluorescent compound, they may also open up new possibilities for its application. ß 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Regulation and measurement of oxidative stress in apoptosis

“…Also, using DAF-2 DA in systems where intracellular Ca2+ increases may also produce misleading results of NO production. Because of these factors, confocal microscopy measurements of DAF-2 DA fluorescence in cells is prone to numerous errors (Broillet et al, 2001). Changes in intracellular Ca2+ occurs in several apoptotic systems (Lopez; Creagh and Donovan).…”

A novel method of measuring nitric-oxide-dependent fluorescence using 4,5-diaminofluorescein (DAF-2) in the isolated Langendorff-perfused rabbit heart

“…One such NO sensitive indicator is 4,5-diaminofluorescein (DAF-2) (Kojima et al, 1998). The membrane-permeable diacetate form (DAF-2 DA) has been used for the detection and imaging of NO from a range of non-cardiac cells and tissues (Broillet et al, 2001;Kashiwagi et al, 2002;Swindle et al, 2004;Kasim et al, 2001;Pittner et al, 2003;Rodriguez et al, 2005). Using flow cytometry, DAF-2 DA has been used in isolated cardiomyocytes (Strijdom et al, 2004) but this method is not possible in the whole heart.…”

Performance of diamino fluorophores for the localization of sources and targets of nitric oxide

“…These new findings add to the growing body of knowledge on how to best optimize their use [9,10] and avoid potential pitfalls [62][63][64][65][66], which will ultimately help investigators to use this relatively new class of fluorophores more judiciously for the investigation of NO-related events.…”