Multiwell Fluorescence Method for Glutathione Determinations in Primary Hepatocytes Using Monochlorobimane

摘要

The fluorophore monochlorobimane (MCB) has been shown to have a relatively high specificity for GSH. Using a fluorescence plate reader with experimental filters, a 380 nm EX filter paired with either a 490 nm or 485 nm EM filter yielded the highest MCB signal-to-noise ratio. The level of GSH in primary hepatocytes was approximately 2-fold higher than that measured in immortalized WB344 rat hepatocytes. The minimum cell number required for measurements of MCB-GSH associated fluorescence was found to be 3 x 10(exp 4). The in vitro dose response study with trimer and tetramer chlorotrifluoroethylene (CTFE) oligomer acids correlated will with previous in vivo toxicity studies. Noncytotoxic exposure of hepatocytes to the tetramer CTFE oligomer acid resulted in decreased cellular GSH levels, while an equivalent dose of the trimer CTFE oligomer acid resulted in elevated cellular GSH levels. The results of this study indicated that intracellular GSH status and fatty acid chain length may play roles in determining the extent of cytotoxicity induced by halogenated fatty acids. Furthermore, it was also found that the newly developed excitation and emission filters could be used to measure intracellular GSH levels using a multiwell plate reader format.