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Anal Methods. 2014 May 7;6(9):3019-3024.

Measurement of DCF fluorescence as a measure of reactive oxygen species in murine islets of Langerhans.

Author information

  • Florida State University, Department of Chemistry and Biochemistry, 95 Chieftain Way, Tallahassee, USA.

Abstract

In islets of Langerhans, oxidative stress induced by reactive oxygen species (ROS) is thought to be critically involved in β-cell dysfunction during the development of diabetes. However, ROS have also been hypothesized to play a role in cellular signalling. To aid in delineating the effects of ROS in living islets of Langerhans, the endocrine portion of the pancreas that contain β-cells, we sought to develop a robust and reproducible protocol to measure these species using the fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA). The protocol that was developed minimized photobleaching and leakage of H2DCF from murineislets and utilized a normalization procedure to further reduce experimental variability. The method allowed for ~25 min of DCF measurement in living islets. We used the developed protocol to compare DCF fluorescence from batches of islets incubated in varying glucose concentrations and observed ~1.5-fold higher fluorescence signals in 3 vs. 20 mM glucose. The effects of diazoxide, which clamps open K+ ATP channels reducing intracellular [Ca2+] ([Ca2+]i) without affecting glucose metabolism, were also investigated. The presence of diazoxide increased DCF fluorescence at all glucose concentrations tested while addition of 30 mM K+ to increase [Ca2+]i reduced the fluorescence by ~15%. With the developed protocol, all experimental methods tested to increase [Ca2+]i resulted in a decrease in DCF fluorescence, potentially indicating involvement of ROS in intracellular signalling cascades.

PMID:
24955137
[PubMed]
PMCID:
PMC4061712
[Available on 2015/5/7]
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