Quantifying cAMP Levels Using HitHunter Assay

Assays were performed as previously described [26 (link)]. Briefly, the cAMP Hunter cells were plated in a 384-well white tissue culture microplate at a 10,000 cells/well density and incubated overnight at 37 °C. Compounds were first dissolved in DMSO to form 5 mM stock solutions, and then 9–10 doses of 100X solutions were prepared by serial dilution with DMSO. Subsequently, these 100X solutions were further diluted with assay buffer consisting of Hanks’s buffered salt solution, HEPES, and forskolin to generate the 5× working solutions. In the agonist assay, cells were treated with compounds (at 1× final concentration) and incubated at 37 °C for 30 min. In the antagonist assay [22 (link)], cells were pretreated with compounds for 15 min at 37 °C followed by 30 min incubation at 37 °C with selected agonists at their EC50 or EC90 dose. The HitHunter cAMP Assay for Small Molecules by Eurofins DiscoverX (Fremont, CA, USA) was then used according to the manufacturer’s directions and the BioTek Synergy H1 hybrid and Cytation 5 plate readers (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 (BioTek, Winooski, VT, USA) were used to quantify luminescence.