Quantitative kinetics of intracellular singlet oxygen generation using a fluorescence probe.

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