Real-Time and High Throughput Monitoring of cAMP in Live Cells Using a Fluorescent Membrane Potential-Sensitive Dye
AbstractAdenosine-3',5'-cyclic monophosphate (cAMP) conveys the signals from G-protein coupled receptors (GPCRs) and regulates a variety of downstream cellular events. However, there are few robust assays available for measuring cAMP in live cells. Most of the existing cAMP assays require cell lysis and/or have relatively low throughput. We report a live cell-based cAMP assay that has been developed to record the real-time changes in intracellular cAMP. By employing a mutated cyclic-nucleotide-gated ion channel (CNGC) as the cAMP biosensor, the change of cAMP level is coupled to the change of transmembrane potential that is measured through a new fluorescent membrane potential (MP)-sensitive dye, HLB 021-152. We have successfully used HLB 021-152 for homogeneously monitoring cAMP stimulations in live cells under both serum-containing and serumfree environments. Upon stimulating the endogenous or heterogenous GPCRs on CNGC-cloned human embryonic kidney 293 cells with agonists, the fluorescence signal of HLB 021-152 increases rapidly. It has much greater assay dynamic range than DiSBAC 2 (3), the existing "gold standard" dye for measuring cellular MP. This new MP dye can be readily formulated for high throughput screening of GPCR modulators either with serum or without serum.