The overlapping fluorescence distributions of AF-12 and ER-Tr prompted us to investigate whether NRB and glibenclamide could compete with their respective fluorescent derivatives in LX2 cells. To this end, LX2 cells were pre-treated with high concentrations (100 μM) of either NRB or glibenclamide, and then loaded with either NRB-AF12 or ER-Tr. Interestingly, pre-treatment with the non-labeled compounds failed to prevent fluorescent labeling following addition of the fluorescent derivatives (data not shown).