Measuring Intracellular cAMP Levels

The levels of the intracellular 3′, 5′-cyclic AMP (cAMP) were measured by the GloSensor cAMP assay (Promega) as described in the previous studies^{28 (link),41 (link)} which has been widely used. The human full-length SSTRs and mutants were cloned into the pcDNA3.1+ vector with a HA signal sequence followed by a Flag tag at the N-terminus. HEK293 cells (ATCC, CRL-1573) were seeded into 6-well microplates at a density of 1.0 × 10⁶ cells/well and cultured for 24 h at 37 °C with 5% CO₂. Cells were then co-transfected with SSTR wild-type or mutants and GloSensor plasmids. After 24 h of transfection, the cells were dissociated with TrypLE™ Express Enzyme (1X) (Thermo Fisher Scientific) and collected by centrifugation at 500 × g for 5 min. Then the collected cells were suspended in Hank’s balanced salt solution [HBSS (Thermo Fisher Scientific)] supplemented with D-fluorescein potassium salt solution (YEASEN), and then the suspension cells were seeded into 96-well plates with 90 μl/well. After incubating at room temperature for 40 min, 5 μM forskolin and ligands diluted in HBSS were added to each well and the plates were incubated at room temperature for 15 min. Fluorescence signals were measured using the Synergy H1 microplate reader (BioTek) and the data presented are means of at least three independent experiments.