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

In islets of Langerhans, oxidative stress induced by reactive oxygen species (ROS) is thought to be critically involved in beta-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 beta-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 similar to 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 similar to 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 similar to 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.