Navigation Links
Gq-coupled receptor assays: a comparison of FLEXstation and FLIPR





Activation of G q-coupled receptors induces the flow of calcium ions out of intracellular compartments into the cytosol via a signaling cascade. Cell signaling through calcium mobilization is responsible for a broad range of cell functions including cell death, proliferation, adhesion and motility. When these processes malfunction, it can lead to disease. Consequently, the functions of Gqcoupled receptors are widely studied, and these proteins are popular drug targets for biotechnology and pharmaceutical companies.

The cell line used in these studies, M1 CHO, is stably transfected with the M1 muscarinic receptor. This transmembrane protein is a Gqcoupled acetylcholine receptor. It is stimulated with carbachol, which leads to the release of calcium into the cytosol, and is inhibited by pirenzepine. Thesefindings demonstrate that a similar range of EC50 and IC50 values for carbachol and pirenzepine, respectively, are obtained with two different fluorescence microplate readers, FlexStation or FLIPR.

Cells were seeded the night before the experiment at a concentration of 30,000 cells/well in a volume of 100 l per well of black walled, clear bottomed, 96-well microplates (E&K distributors, cat# 655090). The next day, cells were incubated for one-half of an hour with 1X Loading Buffer from the FLIPR Calcium Assay Kit. This buffer contained a final concentration of 2.5 mM probenecid to inhibit endogenous efflux pumps present in M1 CHO cells. FlexStation and FLIPR were used to dispense agonist and antago nist into the microplate containing cells and then monitor the mobilization of calcium into the cell cytosol.

Half-log dilutions of activator or inhibitor were used. Specifically, the range of 0 to 1 M for carbachol was used for agonist studies. For antagonist studies, cells were first treated with a range (0 to 10 M) of pirenzepine for a total of two minutes, then a dose of 0.5 M carbachol was added to all wells of the microplate. Results are representative of at least two independent experiments performed over a period of at least two different days. Each point on the dose response curves (Figures 5 and 6) is an average of eight replicates. Each replicate value is the difference between the maximum response to agonist and the minimum or background signal (Max-Min). The curve fit for the dose response curve is 4-parameter, for which the C value is the concentration of drug that causes 50% of maximal response (EC50 or IC50 value, see Figures 5 and 6). The EC50 values for carbachol using FlexStation ranged from 1230 nM, whereas it ranged from 1316 nM using FLIPR. The average IC50 value for pirenzepine was 458 nM using FlexStation and 523 nM using FLIPR. The FlexStation data in Figures 1 and 3 were smoothed using a moving average of three and four points, respectively.

As described above, the EC50 and IC50 values obtained for carbachol and pirenzepine, respectively, were consistent between FlexStation and FLIPR. Since cell preparation and treatment are the same when using either instrument, this allows for assay development on the FlexStation and easy assay transfer to the FLIPR for screening purposes. Other benefits are that SoftMax Pro software makes it simple to set up the instrument and analyz e the data. In conclusion, FlexStation provides an easy-to-use, versatile tool to facilitate assay development.

1 Buck, M. A., and C. M. Fraser. 1990. Biochem. Biophys. Res. Commun. 173: 666-672.

USA 800-635-5577
UK +44-118-944-8000
Germany +49-89-9605-880
Japan +81-6-6399-8211

Check our web site for a current listing of our worldwide distributors.

back to top



Page: All 1 2 3

Related biology technology :

1. Gq-coupled Receptor Assays Using FLEXstation
2. Gq-coupled receptor assays using FLEXstation: M1 muscarinic receptor
3. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System
4. The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System
5. The development of a melanocortin MC5 receptor binding assay using the LEADseeker Multimodality Imaging System
6. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System
7. Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 1000
8. The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR
9. The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies
10. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 3000
11. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System
Post Your Comments:

(Date:11/24/2015)... 24, 2015  Asia-Pacific (APAC) holds the third-largest ... market. The trend of outsourcing to low-cost locations ... higher volume share for the region in the ... margins in the CRO industry will improve. ... ( ), finds that the market earned ...
(Date:11/24/2015)... , Nov. 24, 2015 /CNW/ - iCo Therapeutics ("iCo" ... reported financial results for the quarter ended September ... in Canadian dollars and presented under International Financial ... States ," said Andrew Rae , ... regarding iCo-008 are not only value enriching for ...
(Date:11/24/2015)... -- Clintrax Global, Inc., a worldwide provider of clinical research services headquartered ... the company has set a new quarterly earnings record in Q3 ... posted for Q3 of 2014 to Q3 of 2015.   ... , with the establishment of an Asia-Pacific ... United Kingdom and Mexico , with ...
(Date:11/24/2015)... SHPG ) announced today that Jeff Poulton , Chief ... Annual Healthcare Conference in New York City , ... p.m. GMT). --> SHPG ) announced today that ... Jaffray 27 th Annual Healthcare Conference in New ... 8:30 a.m. EST (1:30 p.m. GMT). --> Shire plc ...
Breaking Biology Technology:
(Date:11/11/2015)... , Nov. 11, 2015   MedNet Solutions , an ... of clinical research, is pleased to announce that it will ... Trials (PCT) event, to be held November 17-19 in ... to view live demonstrations of iMedNet , ... how iMedNet has been able to deliver time ...
(Date:11/10/2015)... , Nov. 10, 2015 ... behavioral biometrics that helps to identify and verify ... Signature is considered as the secure and accurate ... identification of a particular individual because each individual,s ... accurate results especially when dynamic signature of an ...
(Date:11/9/2015)... , Nov. 9, 2015  Synaptics Inc. (NASDAQ: ... solutions, today announced broader entry into the automotive market ... that match the pace of consumer electronics human interface ... sensors are ideal for the automotive industry and will ... Europe , Japan ...
Breaking Biology News(10 mins):