Fig. (3) Intensity of reductide signal mirrors redox-sensitive green fluorescent protein (roGFP).H9c2 cells stably expressing roGFP were seeded into a 96-well plate overnight. The following day, cells were treated with multiple concentrations of NAC, H₂O₂, or vehicle for 60 minutes. Live cell imaging was performed using high-throughput fluorescence microscopy. After image acquisition, cells were washed and reductide was added to each well at a concentration of 1 µM. Cells were incubated for 30 minutes followed by plate reader fluorescence detection (excitation/emission = 485 nm/528 nm). Fluorescence microscopy images following excitation at 405 nm and 488 nm were analyzed using Image J to determine the average ratio of emission intensities for each well. These ratios were compared well by well with the FAM signal from reductide incubation to determine the correlation between reductide and roGFP assessment of redox state. Representative fluorescence microscopy images of H9c2 cells expressing roGFP following excitation at 405 nm and 488 nm are shown (A and B). A ratiometric image following background subtraction is also shown (C). FAM emission signal following incubation of cells with reductide correlated with the concentration of redox modifying agent (NAC or H₂O₂) used to pretreat cells (D and E). RoGFP ratios correlated with the concentration of H2O2 used to pretreat cells but not with NAC (G and H). FAM emission intensity following incubation with reductide was significantly correlated with roGFP ratio following pretreatment with H₂O₂ but not NAC (F and I).