KINETIC-ANALYSIS OF GLUTATHIONE IN ANCHORED CELLS WITH MONOCHLOROBIMANE

A method for the measurement of intracellular glutathione content and glutathione S-transferase activity with monochlorobimane in adherent c ells is described. The method involves the kinetic analysis of monochl orobimane conjugation to glutathione over a relatively short period of time, This permits extrapolation over time for determination of equil ibrium fluorescence intensity (relative glutathione level) from scan i ntensity data that follows first-order kinetics, minimizing problems c ommonly associated with the use of monochlorobimane. By using measured fluorescence intensity values from glutathione standards, a suspensio n calibration curve was generated and, subsequently, was used to deter mine the photomultiplier tube saturation rate, A theoretical intracell ular calibration curve was then generated to quantify glutathione cont ent in cells, This method was also applied to study the changes in glu tathione in a variety of rodent and human cell lines and in selected c ocultures of cells exhibiting similar or different glutathione levels. Comparison of the glutathione levels obtained with monochlorobimane a nd a standard colorimetric method (GSH-400) indicated good correlation between the two methods, These studies support the use of laser cytom etry for measuring intracellular glutathione with monochlorobimane as well as changes in glutathione occurring in cells that establish physi cal contacts with other cells, Laser cytometric analysis of glutathion e in anchored cells also provides opportunities to monitor individual cellular responses to a variety of experimental manipulations, such as responses to various toxic insults or the protective effects of gap j unction-mediated intercellular communication. (C) 1995 Wiley-Liss, Inc .