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.