A multiplex calcium assay for identification of GPCR agonists and antagonists.
Activation of G(q) protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1,536-well plate format for high-throughput screening (HTS). Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a G protein-coupled receptor (GPCR). This multiplexed assay format provides an efficient and cost-effective method for HTS of G(q)-coupled GPCR targets.
Liu
Ke
K
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland, USA.
Southall
Noel
N
Titus
Steve A
SA
Inglese
James
J
Eskay
Robert L
RL
Shinn
Paul
P
Austin
Christopher P
CP
Heilig
Markus A
MA
Zheng
Wei
W
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002138:Calibration; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007700:Kinetics; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D011869:Radioligand Assay; D043643:Receptor, Muscarinic M1 / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000037:antagonists & inhibitors*
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; C484532:NPSR1 protein, human; D043643:Receptor, Muscarinic M1; D043562:Receptors, G-Protein-Coupled; D000242:Cyclic AMP; D002118:Calcium