pTAS2R20 functional assay
HEK-293T cells were seeded at a density of 3×103 cells per well into 96-well black-wall, clear-bottom microtiter plates (Corning, USA). After 24 h of adherent culture, the vectors of the bitter receptor, mG15 and G-CaMP were transiently cotransfected into HEK-293T cells (Behrens et al., 2017). After 36 h, the cells were stimulated with the optimal activation concentration of bitter compounds dissolved in serum-free medium and DMSO, not exceeding a final concentration of 1% (v/v). The activation of the bitter receptors led to the release of Ca2+ from the endoplasmic reticulum and an increase in the concentration of cytoplasmic Ca2+ which was detected with the calcium indicator G-CaMP. Fluorescence images were recorded every 3 s within a period of 30 s before and 120 s after the addition of bitter compounds. The fluorescence values of the cells at each time point were measured with ImageJ software. We counted at least 20 cells in every group and calculated the changes in their fluorescence values compared to the initial stage. The response curves were corrected according to the background fluorescence and normalized to the maximal response observed. The formula for calculating the ratio of the cell fluorescence increase compared to the initial value was as follows:
F = (Fx - Fcx)/(F0 - Fc0)
where F is the fold change of the fluorescence value, Fo is the average fluorescence intensity of the cells before adding the agonists at the first three time points, FC0 and FCX are the background fluorescence values, and Fx is the fluorescence intensity of the cells recorded every 3 s.