Jump to main content
Jump to site search

Issue 9, 2014
Previous Article Next Article

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

Author affiliations

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 murine islets 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.

Graphical abstract: Measurement of DCF fluorescence as a measure of reactive oxygen species in murine islets of Langerhans

Back to tab navigation

Publication details

The article was received on 03 Feb 2014, accepted on 15 Mar 2014 and first published on 18 Mar 2014


Article type: Paper
DOI: 10.1039/C4AY00288A
Author version available: Download Author version (PDF)
Citation: Anal. Methods, 2014,6, 3019-3024
  •   Request permissions

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

    X. Wang and M. G. Roper, Anal. Methods, 2014, 6, 3019
    DOI: 10.1039/C4AY00288A

Search articles by author

Spotlight

Advertisements