Real-time monitoring of genetically encoded redox probes in mammalian cell monolayers
The oxidation state of a cell can shed light on pathologic events. The fluorescent dye roGFP can be genetically fused to thiol peroxide (ORP1) serving as a genetically encoded redox sensitive probe. More specifically, the excitation maxima of roGFP-ORP1 change with redox state. Fully oxidized the fluorophore is maximally excited at a wavelength of 488 nm whereas the completely reduced molecule is excited at 405 nm, while both emit at 510 nm.
The PHERAstar® meets the requirement of a highly sensitive instrument that is needed to measure roGFP. This was shown in lung adenocarcinoma cells expressing the sensor. They responded to H2O2 with an increase or decrease in fluorescence emission at 510 nm, if excited at 400 nm or 485 nm, respectively. Data processing enabled to determine the degree of oxidation and the kinetic of oxidation was determined after addition of H2O2 or a pharmacological compound.