Fig. (1) Reductide fluorescence intensity depends on reduced and oxidized glutathione concentrations in acellular buffer.Reductide was dissolved in TBS pH 7.4 containing reduced glutathione (GSH) +/- oxidized glutathione (GSSG). In samples containing GSH and GSSG, the total glutathione pool (reduced glutathione plus two times oxidized glutathione) was 5 mM. Reductide concentration was 1 µM in all samples. Buffer was assayed in a fluorescence plate reader for FAM (485 nm excitation/528 nm emission) and TAMRA (530 nm excitation/590 nm emission). A. FAM emission intensity from TBS buffer prepared with reduced GSH. B. In the presence of GSH and GSSG, FAM emission intensity increases over time and in proportion to the GSH/GSSG ratio. The addition of GSSG to the buffer results in slower development of FAM signal and reduced maximal FAM emission intensity in comparison with buffer containing only GSH. Intensity of TAMRA emission is also dependent on GSH concentration (C) and GSH/GSSG ratio (D). Unlike FAM, TAMRA emission is insensitive to time (panels C and D).