Quantitative kinetics of intracellular singlet oxygen generation using a fluorescence probe

Singlet oxygen ((1)O(2)) is a type of reactive oxygen species involved in numerous physiological activities. We previously reported that (1)O(2)-specific oxidation products are increased in patients with prediabetes, suggesting that measurement of (1)O(2) may be an important indicator of physiological and pathological conditions. The turnover in the generation and quenching of (1)O(2) is extremely rapid during biological activities owing to it high reactivity and short lifetime in solution. However, the dynamic changes in (1)O(2) generation in living cells have not been fully explored. In this study, we investigated whether the kinetics of (1)O(2) generation can be quantified using a far-red fluorescent probe for mitochondrial (1)O(2), Si-DMA, following addition of the (1)O(2) generator, endoperoxide, to mammalian cells. The kinetics of Si-DMA fluorescence intensity dose-dependently increased following treatment of mammalian living cells with endoperoxide. Alternatively, treatment with (1)O(2) quenchers decreased the fluorescence intensities following endoperoxide treatment. Our results indicate that the kinetics of intracellular (1)O(2) can be readily obtained using Si-DMA and time-lapse imaging, which provides new insights into the mechanism of (1)O(2) generation in mammalian cells and the exploration of (1)O(2) generators and quenchers.