Characterization of RBC of roGFP2 transgenic mice. (A) FACS analysis of RBC from a roGFP2 transgenic mouse and a nontransgenic littermate (WT) demonstrates that ∼ 55% of peripheral RBC express roGFP2. Histogram is representative of 4 experiments using a total of 20 mice. (B) Survival curves of GFP(+) and GFP(-) RBC are identical. Peripheral RBCs of transgenic mice were biotinylated and the percentage of Biotin(+) GFP(+) and Biotin(+) GFP(-) cells was tracked by FACS (mean ± SEM; N = 12). (C) RoGFP2 ratio in RBC treated with the indicated concentration of tBOOH (mean ± SEM from 5 independent experiments). (D) Real-time recording of dynamic and reversible changes in the roGFP2 sensor. RoGFP2 expressing RBC were treated with 1μM H₂O₂ or 10μM tBOOH for 15 minutes, followed by administration of 10mM DTT, all at room temperature. The ratio 405/488 was recorded by continuous flow cytometry. (E) The relationship between roGFP2 ratio and the degree of roGFP2 oxidation (OxD_roGFP2). For maximal reduction, RBCs were treated with 10mM DTT; oxidation was measured over a range of tBOOH concentrations (0.1μM to 100μM). Fluorescence intensity was measured by FACS to define fully reduced (I_488red), fully oxidized (I_488ox) and intermediate states of roGFP2. The OxD_roGFP2 was calculated according to Equation 1, and plotted versus the 405/488 ratio. (F) The relationship between R405/488nm and the roGFP2 redox potentials. The roGFP2 redox potentials were calculated according to the known OxD_roGFP2 in panel E and Equation 2, and then plotted against ratio 405/488nm. GFP emission was recorded for both excitation wavelengths (405 and 488 nm) and is shown as the ratio of 405/488.