FIGURE 2.

Calcium responses of Tas2r-expressing cells challenged with different bitter compounds. A, for calcium imaging experiments, cDNA constructs coding for the indicated Tas2r or empty vector (pcDNA5 or pEAK10 = MOCK) were expressed in HEK293T-Gα16gust44 cells, seeded in 96-well microtiter plates. Calcium traces were recorded in an automated fluorescence imaging plate reader (FLIPR^tetra) based on the detection of changes in fluorescence after the application of the indicated bitter compounds (first stimulus). A second application of 100 nm somatostatin-14 activating endogenous somatostatin receptors served as vitality control. Arrows point to cellular responses specific to bitter compound stimulation. Specificity of receptor activation was controlled by application of same substances on mock-transfected cells. Furthermore, the reliability of the experiment was checked every time by running parallel assays for human TAS2R with known activation patterns (right columns). B, exemplary magnified calcium traces for indicated bitter receptors and mock-transfected cells after application of denatonium saccharide (first stimulus). To demonstrate cell vitality a second application with a final concentration of 100 nm somatostatin-14 was included to activate endogenous somatostatin receptors.