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Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide.
Glia. 2002 Apr 15; 38(2):103-14.GLIA

Abstract

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-gamma (IFN-gamma). 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 microM. 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(-*)(2) in a cell-free solution. The fluorescence due to NO was indeed larger when O(-*)(2) 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.

Authors+Show Affiliations

Otto-von-Guericke University, Institute for Medical Neurobiology, Magdeburg, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

11948804

Citation

Roychowdhury, Sanjoy, et al. "Oxidative Stress in Glial Cultures: Detection By DAF-2 Fluorescence Used as a Tool to Measure Peroxynitrite Rather Than Nitric Oxide." Glia, vol. 38, no. 2, 2002, pp. 103-14.
Roychowdhury S, Luthe A, Keilhoff G, et al. Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide. Glia. 2002;38(2):103-14.
Roychowdhury, S., Luthe, A., Keilhoff, G., Wolf, G., & Horn, T. F. (2002). Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide. Glia, 38(2), 103-14.
Roychowdhury S, et al. Oxidative Stress in Glial Cultures: Detection By DAF-2 Fluorescence Used as a Tool to Measure Peroxynitrite Rather Than Nitric Oxide. Glia. 2002 Apr 15;38(2):103-14. PubMed PMID: 11948804.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Oxidative stress in glial cultures: detection by DAF-2 fluorescence used as a tool to measure peroxynitrite rather than nitric oxide. AU - Roychowdhury,Sanjoy, AU - Luthe,Andreas, AU - Keilhoff,Gerburg, AU - Wolf,Gerald, AU - Horn,Thomas F W, PY - 2002/4/12/pubmed PY - 2002/5/31/medline PY - 2002/4/12/entrez SP - 103 EP - 14 JF - Glia JO - Glia VL - 38 IS - 2 N2 - 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-gamma (IFN-gamma). 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 microM. 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(-*)(2) in a cell-free solution. The fluorescence due to NO was indeed larger when O(-*)(2) 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. SN - 0894-1491 UR - https://www.unboundmedicine.com/medline/citation/11948804/Oxidative_stress_in_glial_cultures:_detection_by_DAF_2_fluorescence_used_as_a_tool_to_measure_peroxynitrite_rather_than_nitric_oxide_ L2 - https://doi.org/10.1002/glia.10024 DB - PRIME DP - Unbound Medicine ER -