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



FLEXSTATION IS A FLEXIBLE TOOL FOR ASSAY DEVELOPMENT AND TRANSFER TO FLIPR

SIMPLE, FAST EXPERIMENTAL SET-UP

EASY DATA ANALYSIS

COMPARABLE EC50 AND IC50 VALUES USING FLEXSTATION OR FLIPR


Introduction
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.


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). 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 antagonist into the microplate containing cells and then monitor the mobilization of calcium into the cell cytosol.


DETERMINATION OF EC50 AND IC50 VALUES
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.



SUMMARY
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 analyze 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.


SALES OFFICES
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.
www.moleculardevices.com


back to top


'"/>

Source:


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:
*Name:
*Comment:
*Email:
TAG: coupled receptor assays comparison FLEXstation and FLIPR

(Date:7/29/2015)... 2015 Sanofi, a global ... second quarter of 2015. CEO Olivier Brandicourt ... performance in different businesses. Watch video ... Topics covered in the interview ... - Performance drivers - Diabetes ...
(Date:7/29/2015)... /PRNewswire/ - BioAmber Inc. (NYSE: BIOA ) today announced ... Officer has resigned.  The Company has hired Andrew ... in December 2014, as full time interim CFO and ... Andy Ashworth began his employment today, ... transition.  Andy was the Company,s CFO from September 2011 ...
(Date:7/29/2015)... Wis. (PRWEB) , ... July 29, 2015 , ... ... identification solutions, today announced its GHS Label Guide . To align chemical ... System (GHS), this guide includes explanations of label components, an example of an ...
(Date:7/29/2015)... ... 2015 , ... The third Medical Innovation Impact Index (MI3) Alert considers FDA ... combinations. The current system received a score of 0 (range -10 to +10) for ... of Fairleigh Dickinson University’s ( FDU ) Rothman Institute of Innovation and Entrepreneurship. ...
Breaking Biology Technology:BioAmber Announces Change in Chief Financial Officer 2BioAmber Announces Change in Chief Financial Officer 3Brady Announces a New GHS Label Guide 2Third Medical Innovation Impact Index Issued by Fairleigh Dickinson University’s Initiative for Patient-Centered Innovation (iPCI) 2Third Medical Innovation Impact Index Issued by Fairleigh Dickinson University’s Initiative for Patient-Centered Innovation (iPCI) 3
... been given a new Web-based tool that makes it ... graduates and students of Wisconsins universities and colleges. , ... career services offices at the University of Wisconsin-Madison, and ... other four-year campuses of the UW System, the Wisconsin ...
... recently participated in a roundtable discussion with HR executives ... talent management and resourcing strategies for elite multinational firms. ... how to make the work place and the workforce ... to make sure that companies always have the right ...
... reexamine the federal policy on stem cell research. ... this debate, because our state is the birthplace of human ... Thomson at the University of Wisconsin-Madison announced that his ... stem cells from human embryos and grown them in five ...
Cached Biology Technology:New Web site makes recruiting from Wisconsin universities easier 2Stuck in Talent Gridlock 2Stuck in Talent Gridlock 3Gov. Doyle comments on unlocking the power of power of stem cell research 2Gov. Doyle comments on unlocking the power of power of stem cell research 3
(Date:7/31/2015)... julho de 2015 A 10 a. ... realizada pela BGI de 22-25 de outubro de 2015, em ... A conferência está celebrando seu 10 o. aniversário este ... tornou uma das reuniões mais influentes do mundo no ... dinâmicos, entusiastas e prazerosos. A ICG-10 ...
(Date:7/31/2015)... The 10 th International Conference on Genomics (ICG-10, ... in Shenzhen , China ... anniversary this year. Since its inauguration in 2006, ICG ... in the ,omics, fields, and is one of the most ... focuses on recent breakthroughs and advances in ,omics, research that ...
(Date:7/23/2015)... July 23, 2015 Research and Markets ... the "Global Outlook of the Biometrics Industry ... The global biometrics market is likely ... commercial applications owing to the uptake of multiple ... transformation that enhances the growth of biometrics in ...
Breaking Biology News(10 mins):A 10a. Conferência Internacional sobre Genômica (ICG-10) será em outubro 2A 10a. Conferência Internacional sobre Genômica (ICG-10) será em outubro 3The 10th International Conference on Genomics (ICG-10) to Open in October 2Global Outlook of the Biometrics Industry 2015 2Global Outlook of the Biometrics Industry 2015 3
... to the lab to understand the fundamental combustion science ... scientific attention for years, both on Earth and in ... and Suppression of Solids-II (BASS-II) experiments, which recently launched ... cargo resupply mission. Designed by researchers at NASA,s ...
... "mask" the noise of swimmers from sharks after receiving ... Strategy. The project, led by Dr Christine Erbe ... first look at characterising noises produced from swimming, surfing ... large shark species. Researchers will then compare ...
... in healthy foods are invited to a day-long conference ... findings on connections between fruit and vegetable production and ... include presentations by researchers, producers, allied produce industry leaders ... 20th anniversary of Texas A&M University,s Vegetable and Fruit ...
Cached Biology News:Combustion continues to draw researchers to space station 2Combustion continues to draw researchers to space station 3Curtin researchers to hide our splashes from sharks 2US food industry leaders to convene for health talks in Texas 2US food industry leaders to convene for health talks in Texas 3
... of Parkers MX80 family of positioners, offering longer ... hole patterns mimic the MX80 family, allowing users ... brackets to build multi-axis systems., , Miniature ... , Accelerations to 5 gs , Velocities to ...
... is from the C-terminal end of the mouse retinoic acid receptor beta ... to 448 of RAR*. The sequence of PEP-005 is (amino to ... P - S - S - V - E - N - ... P - L - L - Q (The N-terminal cysteine has ...
... Rat Neural Stem Cell Kit with Medium ... (NCSs) complete with optimized base medium for ... plate these unique cells generated from E18 ... need for a tissue-like mixture of in ...
... The potential applications for the use of ... gene expression via the RNA interference (RNAi) ... cellular and molecular biology fields. siRNA for ... either chemically or enzymatically, and then transfected ...
Biology Products: