Biochemistry & Analytical Biochemistry

Abstract

The Use of Dichlorofluorescein as a Probe for Monitoring the Effects of Calcium on ROS Production in Mitochondria

Olga V Akopova, Liudmila Kolchinskaya and Valentina Nosar

Dichlorofluorescein (DCF) is widely applied for monitoring reactive oxygen species (ROS) production in cells and isolated mitochondria. Our purpose was to compare different approaches to the use of DCF for monitoring ROS formation accompanying Ca2+ transport in isolated mitochondria. Ca2+ effects on ROS production was studied in rat brain and liver mitochondria using two modes of DCF application: the first was preloading of stock mitochondrial suspension with DCF precursor dichlorofluorescein diacetate (DCFDA), and the other was the addition of the aliquots of DCFDA directly to the incubation medium. Calcium increased ROS production in brain and liver mitochondria both under steady-state and non-equilibrium conditions caused by the gradual release of cytochrome c and the gating of the electron flow. Using DCF-preloaded mitochondria we have shown that the rate of ROS formation under steady-state conditions was linearly dependent on the rate of Ca2+-cycling and Ca2+-stimulated respiration. Transition to non-equilibrium state resulted in the non-linear rise in ROS formation, which too exhibited dependence on Ca2+ concentration. While in DCF-preloaded mitochondria DCF fluorescence closely followed the time course of Ca2+ transport, in unloaded mitochondria probe loading interfered with the detection of ROS formation because of continuing change of DCF concentration into matrix space. Based on the experiments, we came to the conclusion that DCF preloaded in mitochondria could serve a suitable probe for monitoring the effects of Ca2+ transport on ROS production.