Oxidative stress in glial cultures: Detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide
Article first published online: 6 MAR 2002
Copyright © 2002 Wiley-Liss, Inc.
Volume 38, Issue 2, pages 103–114, 15 April 2002
How to Cite
Roychowdhury, S., Luthe, A., Keilhoff, G., Wolf, G. and Horn, T. F.W. (2002), Oxidative stress in glial cultures: Detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide. Glia, 38: 103–114. doi: 10.1002/glia.10024
- Issue published online: 6 MAR 2002
- Article first published online: 6 MAR 2002
- Manuscript Accepted: 10 OCT 2001
- Manuscript Received: 9 OCT 2001
- Deutsche Forschungsgemeinschaft (DFG)
- Bundesministerium für Bildung. Grant Numbers: NBL-3 01220107, WO 474/11-4, EN 366-2/1
- Sachsen-Anhalt. Grant Number: 2997A/0088G
- nitric oxide;
- confocal microscopy.
4,5-diaminofluorescein diacetate (DAF-2DA) is widely used as a fluorescent probe to detect endogenously produced nitric oxide (NO). Recent reports that refer to the high sensitivity of DAF-2 toward NO prompted us to test its efficiency and specificity in a mixed murine primary glial culture model, in which the NO-synthesizing enzyme inducible nitric oxide synthase (iNOS) is expressed by stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Cultures were loaded with DAF-2DA and the fluorescence was measured using confocal microscopy. NO production in the cultures was determined using the ozone/chemiluminescence technique. Due to the extremely high photosensitivity of DAF-2, low laser intensities were used to avoid artifacts. No difference in DAF-2 fluorescence was observed in NO-producing cultures compared to control cultures, whereas the NO/peroxynitrite-sensitive dye 2,7-dihydrodichlorofluorescein (DCF) showed a significant fluorescence increase specifically in microglia cells. A detectable gain in fluorescence was seen when NO-containing buffer was added to the DAF-2DA–loaded cells with a minimum NO concentration at 7.7 μM. An additional gain of DAF-2 fluorescence was obtained when the cells were depleted of glutathione (GSH) with L-buthionine S,R-sulfoximine (BSO). Hence, we monitored the change in DAF-2 fluorescence intensity in the presence of NO and O in a cell-free solution. The fluorescence due to NO was indeed larger when O was added, implying a higher sensitivity of DAF-2 for peroxynitrite. Nevertheless, our results also indicate that measurement of DCF fluorescence is a better tool for monitoring intracellular changes in the levels of NO and/or peroxynitrite than DAF-2. GLIA 38:103–114, 2002. © 2002 Wiley-Liss, Inc.