Inducing intracellular ros and cellular redox without cell death in mesenchymal cells using microsecondpulsed DBD plasma

Intracellular reactive oxygen species (ROS) is a known activator of cell signaling, however too much ROS causes cell death. Our goal was to determine the optimal microsecond-pulsed Dielectric Barrier Discharge plasma (DBD-plasma) treatment dose (J/cm²) in which ROS/redox responses would potentially activate cell function but not cell death. In this study, we applied DBD-plasma to mesenchymal cells and evaluated intracellular ROS generation, redox, mitochondrial membrane potential and the Live/Dead response to increasing levels of DBD-plasma. Using fluorescent indicators to measure superoxide anion (O₂^-.) (MitoSOX™ Red), reduced glutathione (GSH) (ThiolTracker™ Violet) and mitochondrial membrane potential (ΔΨm) (MitoTracker® Red), we show that DBD-plasma doses of 1-5 J/cm² stimulated an immediate (minutes) increase in O₂^- levels with a concomitant decrease in reduced thiol. The mitosox fluorescence intensity increased with DBD-plasma treatments until 1 J/cm², after which levels plateaued regardless of increased dose. Reduced thiol fluorescent intensity showed an immediate 50% decrease regardless of DBD-plasma treatment dose. A 20% decrease in mitochondrial membrane potential occurred at 2 hrs post-DBD-plasma treatments 1 - 5 J/cm², with significant cell death observed (fluorescent Live/Dead assay; syto- 13/propidium iodide) at levels of 2.5 J/cm² and greater. Taken together, these results provide a range in which DBD (0-2.0 J/cm²) intracellular ROS generation and cellular redox change with little effect on cell viability. Understanding the optimal treatment levels DBD-plasma interactions is essential for developing DBD-plasma treatments to induce directed cell function such as; proliferation, differentiation and targeted gene expression.