Navigation Links
Gq-coupled receptor assays using FLEXstation: M1 muscarinic receptor



FLEXSTATION IS A VERSATILE TOOL FOR ASSAY DEVELOPMENT AND BASIC RESEARCH

SIMPLE, RAPID EXPERIMENTAL SET-UP AND DATA ANALYSIS

DETERMINE EC50 AND IC50 VALUES

EXCELLENT FOR ASSAY DEVELOP-MENT AS DEMONSTRATED BY Z FACTOR


INTRODUCTION

Gq-coupled receptors are transmembrane proteins that are responsible for transmitting extracellular signals to the inside of the cell, which ultimately leads to changes in gene expression. Some Gq-coupled receptors can be activated through the specific binding of ligands or drugs to the extracellular domains of the protein. This interaction leads to downstream events such as mobilization of calcium from intracellular storage vesicles into the cytoplasm of the cell. Improper Gq-coupled receptor function is implicated in cancer, neurodegenerative and cardiovascular diseases. Therefore, it is important to compare receptor function in normal and disease states.

The cell line used in these studies, M1 CHO, is stably transfected with the M1 muscarinic Gqcoupled receptor1. This receptor is stimulated with carbachol, which leads to the release of calcium from the endoplasmic reticulum into the cytosol. Activation of the M1 Gq-coupled receptor can be monitored using FLEXstation in conjunction with either the calcium-sensitive fluorescent dye, Fluo-3, or the FLEXstation Calcium Assay Kit from Molecular Devices. The results demonstrate that a similar range of EC50 values is obtained for carbachol using both detection methods.


METHODS
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). Cells were incubated either for one hour with a buffer containing 4.2 M Fluo-3 or for one-half hour with 1X Loading Buffer from the FLEXstation Calcium Assay Kit (Molecular Devices, cat#R8041) before using FLEXstation to dispense carbachol and monitor the mobilization of calcium into the cell cytosol. All experiments were performed at a final concentration of 2.5 mM probenecid in order to inhibit endogenous efflux pumps present in M1 CHO cells.


DETERMINATION OF EC50 VALUES
Cells were exposed to a range of carbachol (0 to 1 M) using half log dilutions of the agonist. Results are representative of at least four independent experiments performed over a period of at least two different days. The raw data was analyzed as Max-Min and Absolute RFU values (Figures 1, 2). The average value of replicate samples was plotted on a graph using a curve fit of four parameter, for which the C value is the concentration of drug that causes 50% of maximal response (EC50 value, see Figure 3). The EC50 values for carbachol using Fluo-3 ranged from 1939 nM, whereas it ranged from 1230 nM using the FLEXstation Calcium Assay Kit (see Figures 3 and 4).



DETERMINATION OF Z FACTOR
Cells in half of the microplate were treated with 0.5 M carbachol, while cells in the other half of the plate were treated with 1X Reagent Buffer (1X HBSS/20 mM HEPES pH 7.4). The FLEXstation Calcium Assay Kit was used to monitor carbachol-mediated calcium mobilization (Figure 5). Z factor was calculated using the formula2:

The terms sc- and sc+ denote the standard deviation of the negative control and positive control, respectively, whereas |c+ c-| denotes the absolute value of the difference in the mean of the positive control and the mean of the negative control. The Z factor obtained for FLEXstation using the FLEXstation Calcium Assay Kit was 0.65. This indicates a large separation band between the negative and positive controls and an optimized, high quality assay.


SUMMARY
FLEXstation generates highly reproducible results for calcium mobilization assays used to monitor the activity of Gq-coupled receptors (Fig. 3). First, the EC50 values obtained for carbachol were consistent between detection methods, Fluo-3 and the FLEXstation Calcium Assay Kit. Also, assays analyzed on the same day showed less than two-fold differences, and assays analyzed on different days showed less than fourfold differences, which further demonstrates the reliable performance of FLEXstation (data not shown). Lastly, the Z factor determination indicates that FLEXstation provides an excellent tool for assay development for the model system examined. In addition, other benefits include SOFTmax PRO software that makes it simple to set up the instrument and analyze the data. Moreover, the dual monochromators provide increased flexibility for assay development or basic research purposes. In conclusion, FLEXstation provides an easy-to-use, versatile tool to help basic research and assay development laboratories reach their goals.

For more detailed information, please refer to the application note Comparison of FLIPR and FLEXstation for Calcium Mobilization Assays, available for download from Molecular Devices web site at:
www.moleculardevices.com/pages/flex_lit1.html


REFERENCES
1. Buck, M. A., and C. M. Fraser. 1990. Biochem. Biophys. Res. Commun. 173: 666672.
2. Zhang, J-H., T.D.Y. Thomas, and K. R. Oldenburg. 1999. J. Biomol. Screening 4: 6773.

SALES OFFICES
USA 800-635-5577
UK +44-118-944-8000
Germany +49-89-9620-2340
Japan +81-6-6399-8211

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


FLEXstation is a trademark of Molecular Devices Corporation.


back to top


'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Gq-coupled Receptor Assays Using FLEXstation
2. Gq-coupled receptor assays: a comparison of FLEXstation and FLIPR
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:12/24/2014)... , Dec. 24, 2014  United Therapeutics Corporation ... Medtronic, Inc. (NYSE: MDT ) has ... Food and Drug Administration (FDA) for the use ... infusion system (including a newly developed catheter) for ... Injection delivered intravenously to patients with pulmonary arterial ...
(Date:12/24/2014)... 24, 2014   BioLife Solutions , Inc. (NASDAQ: ... marketer of proprietary clinical grade hypothermic storage ... thermal shipping products for cells and tissues  ("BioLife" ... its 2015 Annual Meeting of Stockholders on May 4, ... date for the Annual Meeting represents a change of ...
(Date:12/24/2014)... 2014 Earlier this year in a ... director of the Adult Stem Cell Technology Center, LLC ... and under appreciated unique property of adult tissue stem ... Cells: Misunderstood in the Past, Important for the Future,” ... He gave the address at the 4th World ...
(Date:12/24/2014)... New York (PRWEB) December 23, 2014 According ... the Long Island Affiliate of ITRA Global, the national office ... but steady recovery. This is evidenced by the third ... strong stock market. Low energy costs have held inflation ... The only negative is the housing market remaining soft. , ...
Breaking Biology Technology:United Therapeutics Announces Submission Of Pre-Market Approval Application For Implantable Drug Infusion System To Deliver Remodulin 2United Therapeutics Announces Submission Of Pre-Market Approval Application For Implantable Drug Infusion System To Deliver Remodulin 3United Therapeutics Announces Submission Of Pre-Market Approval Application For Implantable Drug Infusion System To Deliver Remodulin 4BioLife Solutions Sets Date for Annual Meeting of Stockholders 2Adult Stem Cell Technology Center, LLC’s Director Sherley's Address on What’s Holding Back Regenerative Medicine Featured on Omics Group Website 2Adult Stem Cell Technology Center, LLC’s Director Sherley's Address on What’s Holding Back Regenerative Medicine Featured on Omics Group Website 3Adult Stem Cell Technology Center, LLC’s Director Sherley's Address on What’s Holding Back Regenerative Medicine Featured on Omics Group Website 4ITRA Global Reports Long Island Is Out of Sync With The National Office Market 2ITRA Global Reports Long Island Is Out of Sync With The National Office Market 3
... 18 International Group, Ltd.,(Bloomberg: BIG SP, Company ... Conformite Europeenne (CE) Mark approval for its,BioMatrix(R) drug-eluting ... European Union and the countries in Asia and ... drug-eluting stent system,developed internally by the Company, consists ...
... Growth driving second new office opening in North Carolina in last 12 ... ... PARK, N.C., Jan. 17 Quintiles,Transnational Corp. today announced that the global ... is an expansion of Quintiles, clinical research in pharmaceutical,and biotechnology development., ...
... WALDWICK, N.J., Jan. 17 NEXCORE Technology,Inc, a ... licensed all the intellectual property rights for the,development ... endoscopic,surgery. The new system provides significantly greater control ... The patents on the system are held by ...
Cached Biology Technology:Biosensors Receives CE Mark Approval for Its BioMatrix(R) Drug-Eluting Coronary Stent System 2Biosensors Receives CE Mark Approval for Its BioMatrix(R) Drug-Eluting Coronary Stent System 3Biosensors Receives CE Mark Approval for Its BioMatrix(R) Drug-Eluting Coronary Stent System 4Quintiles to Open Clinical Research Office in Wilmington, NC 2NEXCORE Technology Obtains Licenses for Revolutionary Fluid Management System 2
(Date:12/17/2014)... 2014  Automation is fundamentally transforming the travel ... at international borders. Over the past decade, ePassports, ... veteran travelers to self process through border control ... at an increasing number of airports, seaports, and ... According to Maxine Most , Principal at ...
(Date:12/11/2014)... Dec. 10, 2014  That blood pressure plays a role ... Hypertension – the medical term for high blood pressure – ... and the inflatable cuff that,s used in measuring blood pressure ... there,s nothing new about hypertension, its triggers and its effects. ... the condition and the best ways to treat it. ...
(Date:12/10/2014)... WINSTON-SALEM, N.C. , Dec. 9, 2014  Wake Forest ... medical education building for its School of Medicine. Funding ... a larger capital campaign that will be publicly launched ... will be located in the former 60 series R.J. ... Forest Innovation Quarter. Construction will begin immediately with plans ...
Breaking Biology News(10 mins):Automated Border Control (ABC) Transforms the Global Travel Experience With More Than 2500 ABC eGates and APC Kiosks Deployed In Airports, Seaports, and Land Borders Worldwide 2Research points to need for new approaches to treatment of high blood pressure 2Research points to need for new approaches to treatment of high blood pressure 3Research points to need for new approaches to treatment of high blood pressure 4Wake Forest Baptist to Build New Medical Education Facility In Wake Forest Innovation Quarter 2Wake Forest Baptist to Build New Medical Education Facility In Wake Forest Innovation Quarter 3Wake Forest Baptist to Build New Medical Education Facility In Wake Forest Innovation Quarter 4
... of Medical Toxicology (ACMT) have signed an agreement to collaborate ... (JMT), the official journal of the ACMT, beginning in ... of Pennsylvania Press. The Journal of Medical ... the science and practice of medical toxicology. The quarterly ...
... is a matter of debate. A high-profile study a few ... their carbon from trees and leaves, evidence for a very ... new research from the University of Washington shows this is ... diet for fish and other aquatic life, according to research ...
... presents strong evidence that the "synergistic" effect of early-life ... causes more harm to developing lungs than one or ... scientists at the University of Cincinnati (UC) College of ... levels of traffic-related particles and indoor endotoxin during early ...
Cached Biology News:Springer and the American College of Medical Toxicology to work together 2Fish food fight: Fish don't eat trees after all, says new study 2Fish food fight: Fish don't eat trees after all, says new study 3Exposure to both traffic, indoor pollutants puts some kids at higher risk for asthma later 2
Request Info...
Request Info...
... purification systems provide high speed purification of ... offer increased throughput and reproducible results. The ... mL and KingFisher 96, covering sample volumes ... consists of an instrument, specially designed plastics ...
Automated -80° C sample library for microplates and cryotube racks in SBS format; for integration into automated processes....
Biology Products: