In Situ EPR Detection of Reactive Oxygen Species in Adherent Cells Using Polylysine-Coated Glass Plate

In conventional electron paramagnetic resonance (EPR) experiments, it is a common procedure to use a cell suspension after detachment to measure reactive oxygen species (ROS) generated in the cell. However, to elucidate redox signaling transduction between the cells, the sample should be under cell culture conditions. Here, we propose an in situ EPR detection methodology of ROS produced in adherent cells. A quartz glass plate was coated with poly-l-lysine (PLL) to improve the adhesion efficiency of cultured cells. Using mouse fibroblasts and human malignant epithelial cells (HeLa) on the glass sheet (approximately 6.5 x 10(4) adherent cells per sheet), remarkable increases in the relative EPR signal intensities of spin adducts related to superoxide radicals were observed 1 h after addition of a redox-active compound (pyocyanin) and a spin trap to the culture medium. There was no disturbing effect on the EPR signals with the PLL-coated glass plate alone. The intensity of EPR signals in HeLa cells without exogenous pyocyanin was statistically significantly higher than that of the control, implying intrinsically larger amounts of ROS in cancer cells than those in non-malignant ones. Moreover, an optically transparent holder for supporting the cell-adhered glass substrate enabled both EPR detection of ROS and microscopic observation, where more than 88 % of cell viability was maintained. These findings demonstrated that the proposed in situ EPR methodology would be useful for measuring ROS generated under cell culture conditions.