Journal
ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES
Volume 4, Issue 4, Pages 461-471Publisher
MARY ANN LIEBERT, INC
DOI: 10.1089/adt.2006.4.461
Keywords
-
Funding
- NINDS NIH HHS [R44 NS044641-02] Funding Source: Medline
Ask authors/readers for more resources
Adenosine-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 serum-free 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available