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

Abstract

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 9 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.

Detection of Reactive Oxygen and Nitrogen Species by Electron Paramagnetic Resonance (EPR) Technique

“…One of them is diamagnetic spin trapping by using DMPO which can be used to trap superoxide, hydroxyl, and glutathionyl radicals. NO will not directly react with nitrone-based spin-traps to form spin adducts [ 41 ]. NO is considered a relatively stable radical that can conjugate with spin traps.…”

“…This procedure was used for mouse fibroblasts and human malignant epithelial cells (HeLa) and significantly increased the EPR signal intensities of spin adducts related to superoxide radicals 1 h after the addition of a redox-active compound and a spin trap to the culture medium. These findings were demonstrated to be useful for measuring ROS generated under cell culture conditions [ 41 ]. Also, an EPR microinvasive method developed by Mrakic-Sposta et al [ 57 ] provides direct evidence of the “instantaneous” presence of ROS, returning to absolute concentration levels that correlate with “a posteriori” assays of ROS-induced damage by means of biomarkers.…”

Interrogation of drug effects on HeLa cells by exploiting new AFM mechanical biomarkers

“…We note that poly-L-lysine is a widely used biocompatible material for facilitating the attachment of cells on the substrate for AFM measurement and did not show an obvious inuence on cell viability, as reported by others. 27 AFM imaging and force measurement AFM experiments were performed at room temperature using the Dimension Edge Instrument (Bruker Nano Surfaces, Santa Barbara, USA). So AFM cantilevers (MLCT, Bruker Nano Inc., Camarillo, USA) with a nominal spring constant of 0.03 N m À1 were used.…”

Nanotoxicity of Silver Nanoparticles on HEK293T Cells: A Combined Study Using Biomechanical and Biological Techniques

“…It needs to be pointed out that poly- l -Lysine is a biocompatible material that has been widely used for helping the adhesion of cells on the substrate in the AFM experiments and showed no obvious influence on cell viability. 18 , 40 …”