Using roGFP2 to monitor redox dynamics on in vitro PHZ treatment or during in vivo normal RBC aging. (A) RoGFP2 responses to the RBCs oxidative stress caused phenylhydrazine (PHZ). Transgenic RBCs were treated with 10μM and 50μM PHZ at room temperature for 60 minutes. The ratio 405/488 was determined by FACS. Data were from 3 independent experiments, shown as mean ± SEM. (B) DTT reversed the increase in roGFP2 ratio caused by PHZ treatment. Continuous flow cytometry was used to follow dynamic redox changes in RBCs on exposure to 10μM PHZ for 15 minutes followed by 10mM DTT. (C) RBCs of roGFP2 transgenic mice were biotin labeled and analyzed over time by flow cytometry for GFP and biotin. Shown are representative plots from day 7 and day 45 after labeling. (D) RoGFP2 ratio was monitored by flow cytometry using RBC harvested at intervals up to 49 days after labeling. From day +14 after labeling, there is a progressive increase in oxidation of the roGFP2 sensor. Data are mean ratio ± SEM (n = 23); representative of 4 experiments. *** P < .001.