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:1/23/2015)... 2015 Biofertilizers stimulate microbial activities in ... and phosphorous through the activities in the soil or ... due to the stress on reducing environmental pollution and ... report defines and segments the bio fertilizers market with ...
(Date:1/22/2015)... Md. (PRWEB) January 22, 2015 ... its new UV-1280 multipurpose UV-visible spectrophotometer. Offering wavelength ... high-quality instrument is ideal for applications in a ... life science. , The user-friendly UV-1280 enables intuitive ...
(Date:1/22/2015)... (PRWEB) January 21, 2015 Cambridge Semantics, the ... technology, today announced that 2014 was a record-breaking year across ... capabilities of the Anzo Smart Data Platform and our Smart ... utilize insights from diverse data which led to record growth ...
(Date:1/22/2015)... 22, 2015 Pipette.com has added the ... their comprehensive portfolio of Eppendorf products. , The Eppendorf ... a Centrifuge 5424/5424 R and receive the following:, ... Research plus or Eppendorf Reference 2 , ...
Breaking Biology Technology:Biofertilizers Market Expected to Reach $1,649.7 Million by 2019 - New Report By MarketsandMarkets 2Biofertilizers Market Expected to Reach $1,649.7 Million by 2019 - New Report By MarketsandMarkets 3Biofertilizers Market Expected to Reach $1,649.7 Million by 2019 - New Report By MarketsandMarkets 4Shimadzu’s New Monitored Single-Beam UV-Vis Spectrophotometer Offers Comprehensive Measurement Options in a Compact Body 2Cambridge Semantics Announces Record Results for 2014 2Eppendorf Announces Promotional Bundle Which Includes: Eppendorf Centrifuge 5424, 3-Pack of Pipettes, and Tips - Available Now at Pipette.com 2
... WASHINGTON, Oct. 1 Today a promising young,scholar ... Mexico, accepted,the Manuel Velasco-Suarez Award in Bioethics for ... field of bioethics in Latin America during,the 27th ... Naftolin, a trustee of the Pan American Health ...
... Quarter 2007 Earnings Conference Call Thursday, November 8, 2007, ... 5:00 p.m. U.S. EST, FRAZER, Pa., Oct. 1 ... for 5:00 p.m. EST on Thursday, November 8, 2007. ... November 8, 2007 Schedule, 4:30 p.m. EST Q3 2007 results ...
... Oct. 1 A highly sensitive, easy-to-use,test that ... as the Best New Veterinary Product for Diagnostics ... time PCR test was selected for its,excellence and ... the global community of animal sciences practitioners and,professionals. ...
Cached Biology Technology:Prestigious Bioethics Award for Scholar from Mexico 2BoVir(R) Named Best New Veterinary Diagnostics Product in International Animal Pharm Awards 2BoVir(R) Named Best New Veterinary Diagnostics Product in International Animal Pharm Awards 3
(Date:12/11/2014)... , Dec. 10, 2014  That blood pressure plays a ... while. Hypertension – the medical term for high blood pressure ... 1800s, and the inflatable cuff that,s used in measuring blood ... mean there,s nothing new about hypertension, its triggers and its ...
(Date:12/10/2014)... 2014 You,ve been here before: you desperately need to sign ... key or the answer to your secret question. What,s your dog,s ... Hoyos Labs , a digital infrastructure security company, launches ... frustration that comes with usernames, passwords and PINs – 1U ...
(Date:12/10/2014)... WINSTON-SALEM, N.C. , Dec. 9, 2014  Wake Forest Baptist ... building for its School of Medicine. Funding for this $50 ... that will be publicly launched next summer. ... 60 series R.J. Reynolds Tobacco Company complex, adjacent to 525@vine ...
Breaking Biology News(10 mins):Research 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 4The Password is Finally Dead: Launch of 1U Mobile App Eliminates Need for All Usernames and Passwords 2The Password is Finally Dead: Launch of 1U Mobile App Eliminates Need for All Usernames and Passwords 3Wake 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
... field of schizophrenia research has come alive with many ... introduction of chlorpromazine to the current day, all treatments ... had, at their core, a single treatment mechanism, the ... of clozapine in the 1980s suggested a potential that ...
... Loss of a small portion of chromosome 16, known ... from the University of Chicago Medical Center, the University ... Institute in an article, published Dec. 21, 2007, online ... this genetic "microdeletion" occurred in only four out of ...
... Dr. McWhorter Joins Olive, Edwards & Cooper with Technical, Products ... ... Edwards & Cooper,announced the addition of Dr. David McWhorter as a ... expand into the,homeland security marketplace. His immediate focus will be ensuring ...
Cached Biology News:New treatment mechanisms for schizophrenia 2New treatment mechanisms for schizophrenia 3Recurrent genetic deletion linked to autism 2Chemist Brings SAFETY Act Expertise to Homeland Security Firm 2
BD FastImmune EDTA Solution. Applications: Flow Cytometry Storage Temperature: Refrigerate(2 to 8C)...
Autoclavable, variable volumefitted w./ an anti-drip safety valve; borosilicate glass barrelprotected with transparentpolypropylene sleeve; spring-lock cursor design ensuresfine adjustment for exact ...
...
... Detection Platform combines robust chemistry with ... sensitive, specific, and cost-effective detection method ... Platform offers exceptional performance with specificity ... high signal/background ratio, and an expanded ...
Biology Products: