Flow Cytometric Analysis of Intracellular ROS and RNS Production and Curcumin Inhibition

Reactive oxygen species (ROS) and reactive nitrogen species (NOS) are important biological regulators involved in cell damages and health problems. Curcumin, a naturally occurring phenolic compound, has long been recognized as a promising anticancer medicine because it can effectively inhibit ROS generation. However, ROS is a mixture of oxidative species, and the effects of curcumin on different oxidative molecules are unclear. Moreover, ROS analysis via flow cytometry is challenging due to the lack of specific probes for selective detection of ROS and RNS species. To address these issues, we have developed a few novel ROS and RNS probes with high selectivity targeting different ROS and RNS species. Human immortalized T lymphocyte Jurkat cells were loaded with: 1) ROS Green for measuring total ROS; 2) MitoROS 580 for detecting superoxide; 3) Nitrixyte Orange for targeting nitric oxide and 4) DAX-J2 PON Green for probing peroxynitrite, respectively. After 1 hour staining, cells were incubated with various oxidizing agents to trigger specific ROS or RNS. Results of flow cytometric analysis indicated that in the absence of curcumin treatment, fluorescence signal increased significantly in cells upon stimulation with oxidizing agents. In contrast, the addition of curcumin remarkably decreased intracellular total ROS, superoxide and peroxynitrite levels in a dose-dependent manner. The results demonstrated high selectivity of these new probes toward target ROS or RNS over other competing species, as well as good applicability of them for real-time quantitative monitoring of intracellular ROS and RNS production and curcumin inhibition via flow cytometer. In conclusion, we provide a robust method for monitoring different ROS and RNS in live cells and screening potential antioxidant agents for the treatment of various ROS and RNS-related diseases.