Figure 1
Human DP2 receptor conformation sensor suited for measurements in microtiter plates. (A) FRET measurements were performed in 96-well plates containing HEK293 cells stably expressing the DP2 receptor sensor using a Spark 20 M (Tecan) plate reader. (B) Left: depicted is a representative example of the time course of mTurq2 and eYFP emission upon excitation at 430 nm in a single well (corrected for buffer wells, normalized to the three basal cycles). Right: the corresponding alteration in the emission ratio was plotted as Δ(eYFP/mTurq2) of a representative example single well measurement upon addition of 5 µM PGD2. (C) Averaged Δ(eYFP/mTurq2) signals were plotted from experiments similar as described in (B) where a test concentration of PGD2 was applied at cycle 3 followed by reference concentration of 10 µM PGD2 at cycle 20 (final concentration). (D) Each agonist-evoked amplitude of the tested PGD2 concentrations was normalized to the amplitude of the reference concentration. The panel shows the mean ± SEM of the DP2 receptor sensor activation from all experiments and a curve fit to the pooled data (for illustration purposes only). (E) The panel shows pEC50 for three individual experiments. (F) Δ (eYFP/mTurq2) changes in percent of an example plate used to determine the Z-factor of DP2 receptor sensor are plotted over time. Control wells are shown in black (buffer application) and sample wells in red (PGD2 application), n = 30 per group. (G) Z-factor values of three independent DP2 receptor sensor experiments are plotted. Individual experiments are presented in Fig. 1S. (C–G) Data are presented as mean ± SEM.
