Live-cell Assay to Interrogate GPCRs

In a previous post we discussed the advantages associated with luminescent assays mentioning a recently developed biosensor utilized in a live-cell assay format. The ability to either stably or transiently transform the biosensor into a cell line allows investigation of both endogenous and over-expressed GPCR targets. More recently we have demonstrated the ability to miniaturize the assay to use as little as 1,000 cells in a 384-well format to monitor the response of the endogenous β-adrenergic receptor in HEK293 cells (Figure 1).

















The assay conditions were further optimize to determine the optimal reagent equilibration incubation period and both agonist and antagonist incubation time. The Synergy H4 (BioTek Instruments, INC.) was used to measure the luminescence signal generated to determine the EC50 and IC50 of several agonists and an antagonist (Figure 2). The data was presented at ELRIG and MIPTEC conferences earlier this month.















Further efforts demonstrate that the automation of the assay, using the MultiFlo and Precision instruments, will be of particular importance for secondary screening efforts that employ the use of serial dilution of compounds that registered as hits in a screening campaign necessary to construct dose-response curves and determine potency. Assay performance as well as the pharmacology of several compounds were investigated and compared to those determined when manual methods were employed. Two new application notes will be available describing this work on the BioTek website in October.


By, BioTek Instruments