Quantitative kinetics of intracellular singlet oxygen generation using a fluorescence probe

Singlet oxygen (¹O₂) is a type of reactive oxygen species involved in numerous physiological activities. We previously reported that ¹O₂-specific oxidation products are increased in patients with prediabetes, suggesting that measurement of ¹O₂ may be an important indicator of physiological and pathological conditions. The turnover in the generation and quenching of ¹O₂ is extremely rapid during biological activities owing to it high reactivity and short lifetime in solution. However, the dynamic changes in ¹O₂ generation in living cells have not been fully explored. In this study, we investigated whether the kinetics of ¹O₂ generation can be quantified using a far-red fluorescent probe for mitochondrial ¹O₂, Si-DMA, following addition of the ¹O₂ 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 ¹O₂ quenchers decreased the fluorescence intensities following endoperoxide treatment. Our results indicate that the kinetics of intracellular ¹O₂ can be readily obtained using Si-DMA and time-lapse imaging, which provides new insights into the mechanism of ¹O₂ generation in mammalian cells and the exploration of ¹O₂ generators and quenchers.