Journal of Agricultural and Food Chemistry ARTICLE Figure 1. Comparison of the signals obtained using the luminescence assay vs the ﬂuorescence assay. (A) The ﬂuorescence intensities obtained using the ﬂuo-4 assay when the hT1R2/hT1R3-expressing cells were stimulated with 5 mM aspartame (n = 6). (B) The luminescence intensities obtained using the aequorin assay when the hT1R2/hT1R3-expressing cells were stimulated with 5 mM aspartame (n = 6). (C and D) Dose response curve of hT1R2/hT1R3-expressing cells to aspartame using the ﬂuo-4 assay (C) or the aequorin assay (D). Each point on the dose response curves indicates the mean ( SEM (n = 6). ’ MATERIALS AND METHODS Materials. Samples were obtained from commercial sources: Aspar- tame and riboflavin were purchased from Wako Chemical Co. (Osaka, Japan), BCECF [20,70-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein] was from Invitrogen (Carlsbad, CA), coelenterazine was from Promega (Madison, WI), and its derivatives were from Sigma Aldrich (St. Louis, MO). The buffer for the fluorescence assay was composed of 10 mM 4-(2-hydro- xyethyl)-1-piperazineethanesulfonic acid (HEPES), 130 mM NaCl, 10 mM glucose, 5 mM KCl, 2 mM CaCl2, and 1.2 mM MgCl2 (pH adjusted to 7.4 using NaOH). The assay buffer for the luminescence assay was com- posed of the fluorescence assay buffer with the addition of 0.1% bovine serum albumin (BSA, Sigma Aldrich). The ligands were diluted into each of the assay buffers at the desired concentrations. Construction of the Apophotoprotein Expression Plas- mids. The nucleotide sequences of the cDNA fragments encoding the apophotoproteins used were codon-optimized for expression in mam- malian cells using OptimumGene (GenScript), and the optimized cDNAs were chemically synthesized by GenScript Inc. (Piscataway, NJ). The Kozak-consensus sequence was introduced upstream of the start codon for efficient translation. The apophotoproteins were targeted to mitochondria by fusing the nucleotide sequence encoding the first 29 amino acids of the N-terminal leader sequence of cytochrome c oxidase (subunit VIII) immediately before the ATG triplet coding for the initiator Met in each apophotoprotein-coding sequence. The constructs containing the apophotoprotein-coding sequences were subcloned into the Asc I-Not I site of the pEAK10 expression vector (Edge Biosystems, Gaithersburg, MD). Culture of Cells Expressing the Human Sweet Taste Receptor. Flp-In 293 cells stably expressing the human sweet taste receptor, hT1R2/hT1R3, together with the chimeric G-protein, hGα16- gust44, were constructed as previously reported.26 The cells were maintained in low glucose concentration (1.0 g/L) Dulbecco's modified Eagle's medium (DMEM, Sigma Aldrich) supplemented with 10% fetal bovine serum (Invitrogen) at 37 C under 5% CO2. Fluorescence Assay. The cells were seeded at a density of 90000 cells/well into 96-well black-walled CellBIND surface plates (Corning, NY) and cultured at 37 C under 5% CO2. After a 24 h of incubation, the cells were washed with assay buffer, loaded with 5 μM fluo-4AM (Dojindo Laboratories, Kumamoto, Japan) in assay buffer for 30 min at 27 C in the dark, and washed again with assay buffer. Changes in fluorescence intensity (excitation at 485 nm, emission at 525 nm, and cutoff at 515 nm) were monitored using a FlexStation 3 microplate reader (Molecular Devices Co., Sunnyvale, CA) at 2 s intervals. After 20 s of baseline reading, an aliquot of assay buffer supplemented with 2 ligand was added, and the scanning was continued for an additional 100 s. The response was expressed as the ΔRFU (delta relative fluorescence units) calculated as the difference between the maximum and the minimum fluorescence values. The responses were averaged from at least three wells receiving the same stimulus. The signal/ background ratios and EC50 values for the ligand receptor interactions were determined from the concentration response curves generated using Clampfit ver. 9.2.0.09 (Molecular Devices) by fitting the data to the Hill's equation. The signal/background ratio was determined as the maximum signal/minimum signal. Luminescence Assay. Human sweet taste receptor-expressing cells were seeded in six-well culture plates and transfected with 4 μg of the apophotoprotein expression plasmid using Lipofectamine 2000 regent (Invitrogen). After 6 h, the transfected cells were trypsinized, seeded in 96-well black-walled CellBIND surface plates at a density of 100000 cells/well, and cultured overnight at 37 C in 5% CO2. After overnight culture, the medium was removed and replaced with coelen- terazine loading buffer (luminescence assay buffer containing 10 μM coelenterazine, pH 7.4) for 4 h at 27 C in the dark. After 20 s of baseline reading, an aliquot of the luminescence assay buffer supplemented with 2 ligand was added, and the light emission was recorded using a 12132 dx.doi.org/10.1021/jf2029835 |J. Agric. Food Chem. 2011, 59, 12131–12138