Journal of Agricultural and Food Chemistry ARTICLE Figure 5. Comparison of the mitochondrial clytin-II assay with the ﬂuo-4 assay in the presence of riboﬂavin, a ﬂuorescent food substance. (A) Dose response curve obtained using the mitochondrial clytin-II assay when hT1R2/hT1R3-expressing cells were exposed to aspartame in the presence of the indicated concentrations of BCECF. Each point indicates the mean ( SEM (n = 6). (B and C) Fluorescence intensities obtained using the ﬂuo-4 assay when hT1R2/hT1R3-expressing cells were stimulated with assay buﬀer (B) or 5 mM aspartame (C) in the presence of the indicated concentrations of riboﬂavin (n = 3). (E and F) Luminescence intensities obtained using the mitochondrial clytin-II assay when hT1R2/hT1R3- expressing cells were stimulated with assay buﬀer (E) or 5 mM aspartame (F) in the presence of the indicated concentrations of riboﬂavin (n = 3). (D and G) Dose response curve obtained using the ﬂuo-4 assay (D) or the mitochondrial clytin-II assay (G) when hT1R2/hT1R3-expressing cells were exposed to aspartame in the presence of the indicated concentrations of riboﬂavin. Each point indicates the mean ( SEM (n = 3). The EC50 values were measured as 1.0 mM for aspartame, 0.9 mM for aspartame + 0.25 μg/mL riboﬂavin, 1.3 mM for aspartame + 0.50 μg/mL riboﬂavin, and 1.0 mM for aspartame + 1.0 μg/mL riboﬂavin using the mitochondrial clytin-II assay. monitored the increases in intracellular Ca2+ concentration as accurately as their cytoplasmic counterparts. Both mitochondrial clytin-II- and mitochondrial aequorin-based assays should be suitable for use in sweet taste receptor evaluation systems. We have demonstrated the advantage of the luminescence- based taste evaluation system by measuring the cellular responses to aspartame in the presence of the model ﬂuorescent com- pounds BCECF and riboﬂavin. Because BCECF is widely used as an intracellular pH indicator and provides strong ﬂuorescent signals at low concentration, we expected that BCECF would not introduce nonspeciﬁc cellular responses and would serve as a suitable model ﬂuorescent substance. Actually, when we mea- sured the BCECF signals using the ﬂuo-4 assay, 10 40 nM BCECF produced approximately 4 14-fold higher signals than the maximum signals observed in response to aspartame (Figure 2A,B), and we failed to detect the actual sweet taste 12136 dx.doi.org/10.1021/jf2029835 |J. Agric. Food Chem. 2011, 59, 12131–12138