Dynamic analytical chemistry: a kinetic study of the labeling of normal and age fractionated human erythrocytes with monobromobimane

The application of digital fluorescence imaging microscopy for the analysis of the thiol content and labeling kinetics of normal and age fractionated erythrocytes on a single cell basis is described. Monobromobimane (mBBr), a thiol selective dye, reacts primarily with glutathione, the major thiol compound in erythrocytes. The fluorescence intensity of thiol bound mBBr molecules is about 14-fold higher than the fluorescence intensity of unbound mBBr molecules. The labeling process is monitored in-situ using a slow scan high performance charge coupled device (CCD) camera. A 4-fold cell-to-cell variation is found in the maximum fluorescence intensity of mBBr labeled cells within a large normal population of erythrocytes. This observation is in agreement with previous results obtained for the same cellular system using capillary electrophoresis and laser excited fluorescence detection. In addition, a 2-fold variation is observed in the rate of labeling of the cells with mBBr. Quantitative kinetic measurements of the labeling of age fractionated erythrocytes with mBBr show that the cellular variation in the labeling rate and in the cellular thiol content are related to the cell age.