Abstract
Regenerative medicine has attracted much attention worldwide, but noninvasive diagnostic technologies for transplanting cells have not yet been established. As an indicator of cellular tumorigenesis, an increased generation of reactive oxygen species (ROS) has been well known. In this work, we developed an in-situ electron spin resonance (ESR) detection methodology for quantitatively measuring ROS produced in adherent cells. Mouse fibroblasts (NIH3T3) and human malignant cells (HeLa) were used. The cells were cultured on a poly-L-lysine coated quartz glass plate (3 mm × 45 mm), and then the plate was put on an optically transparent holder which enabled both microscopic observation and ESR measurement under cell culture conditions. At 1 h after addition of a redox-active regent (0.1-mM pyocyanin) and a spin trap (DMPO) to the culture medium, remarkable increases in the relative ESR signal intensities from spin adducts related to superoxide radicals were observed in both NIH3T3 and HeLa cells. In HeLa cells, the ESR signal intensities were approximately 2-times higher than those of NIH3T3 cells, which could imply intrinsically larger amounts of ROS in malignant cells.
