Fig. 5
From: Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides
Quantification of cAMP produced by GPCR activation. A cell line constitutively expressing an octopamine receptor from Drosophila melanogaster (HEK293 – DmOctβ1R) was incubated with increasing concentrations (10− 9 – 10− 4 M) octopamine in a 24 MWP for 30 min at 37 °C. Cells were lyzed by adding ice-cold ethanol. Extracts were lyophilized, reconstituted in H2O and the amount of cAMP was determined with purified Epac1-camps-His6 protein as described before. Excitation was at 430 nm and fluorescence emission was recorded at 475 nm (ECFP) and 530 nm (EYFP). After recording basal fluorescence emission for ECFP and EYFP, samples were added and incubated for 30 min at room temperature. Finally, the EYFP/ECFP ratio (R) of fluorescence emission for each well was calculated and normalized to the ratio of the basal fluorescence emission (R0). Using a calibration curve established with known cAMP concentrations in parallel, the cAMP amount present in each sample was determined. Mean values (± SD) from quadruplicate determinations were plotted against octopamine concentrations. The EC50 value (2.475 × 10− 8 M) of DmOctβ1R was calculated with a four-parameter nonlinear regression analysis using GraphPad Prism v5.04. A representative of three independently performed experiments is shown