Fluorescent estimation of H2O2-induced changes in cell viability and cellular nonprotein thiol level of dissociated rat thymocytes

We have developed a procedure to simultaneously estimate cell viability and the cellular level of nonprotein thiol (presumably glutathione) using two fluorescent dyes, 5-chloromethylfluorescein (5CMF) and ethidium, and rat thymocytes. Diethylmaleate and N-ethylmaleimide reduced, respectively, the intensity of 5CMF fluorescence to 0.23 and 0.1, relative to the control. Incubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, decreased the intensity of 5CMF fluorescence to 0.61. Results indicate that 5CMF fluorescence can be attenuated by agents that decrease the level of cellular nonprotein thiols, suggesting that 5CMF fluorescence is utilized for estimating the level of cellular glutathione. Hydrogen peroxide (10 mu M to 3 mM) reduced the intensity of 5CMF fluorescence in a dose-dependent manner and increased the number of thymocytes stained with ethidium (presumably dead cells or cells with compromised membranes) at concentrations of 300 mu M or greater. Reduction of cellular glutathione level seems to precede cell death in which oxidative stress is involved.