Journal of Agricultural and Food Chemistry ARTICLE Figure 2. Comparison of the aequorin assay with the ﬂuo-4 assay in the presence of BCECF. (A and B) Fluorescence intensities obtained using the ﬂuo- 4 assay when hT1R2/hT1R3-expressing cells were stimulated with assay buﬀer (A) or 5 mM aspartame (B) in the presence of the indicated concentrations of BCECF (n = 3). (D and E) Luminescence intensities obtained using the aequorin assay when hT1R2/hT1R3-expressing cells were stimulated with assay buﬀer (D) or 5 mM aspartame (E) in the presence of the indicated concentrations of BCECF (n = 3 6). (C and F) Dose response curve obtained using the ﬂuo-4 assay (C) or the aequorin assay (F) 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 = 3 6). FlexStation 3 microplate reader for an additional 55 s. The changes in luminescence intensity were monitored every 1.5 s and smoothed using a five-point moving average. The response from each well was expressed as RLU (relative light units) and calculated using the area under the curve (AUC). The responses were averaged from at least three wells receiving the same stimulus. To calculate the EC50 values and the signal/ background ratios, plots of the amplitudes versus concentrations were fitted to the Hill's equation. ’ RESULTS Application of the Luminescence-Based Assay to the Detection of Responses Mediated by the Human Sweet Taste Receptor. The luminescence-based assay was performed using a representative photoprotein, aequorin, to detect the taste receptor response. The results were compared with the fluores- cence-based taste evaluation system using a conventional fluores- cent intracellular Ca2+ indicator, fluo-4, to evaluate whether the luminescence-based assay was applicable as a sweet taste receptor assay. The signals following sweet taste receptor activation in response to application of the artiﬁcial sweetener aspartame were measured using both assay systems. As a result, we succeeded in detecting sweetener-induced signaling using the luminescence- based assay; when the receptor was activated by aspartame, the luminescent intensity increased gradually and then declined over time in the same way as the ﬂuorescent intensity monitored using the ﬂuo-4 assay (Figure 1A,B). Moreover, the dose dependency of the response to aspartame was clearly observed using the 12133 dx.doi.org/10.1021/jf2029835 |J. Agric. Food Chem. 2011, 59, 12131–12138