cAMP accumulation assay

The CHO-K1 cells contained human GPR119 under the control of a doxycycline inducible system and a beta-lactamase reporter gene under the control of a cAMP response element (CRE). Addition of doxycycline to these cells allowed for GPR119 expression and a subsequent assay for GPR119 specific activity. Cells were seeded in 384-well plates at a density of 10⁴ cells/well and doxycycline (1 ug/mL) was added to induce GPR119 expression. Cells were treated with compounds at varying concentrations. After 5 h incubation, cellular cAMP levels were measured following the manufacturer’s instructions (Invitrogen). The assay technology is based on fluorescent detection of beta-lactamase reporter gene activity using a fluorescence resonance energy transfer (FRET)-enabled substrate that generates a ratiometric cAMP response. Three replicates for each concentration of compounds were used.