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 antago nist 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 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.


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:


(Date:4/27/2017)... California (PRWEB) , ... April 27, 2017 , ... ... new infographic explaining why mass flow controllers based on capillary thermal mass flow ... industrial gas mass flow control applications. Over 80% of all industrial processes—such ...
(Date:4/27/2017)... -- Pendant Biosciences, Inc. (formerly Nanoferix, Inc.), a privately-held advanced ... technologies, today announced that it has been accepted into ... . Shawn Glinter , Founder ... are excited to become part of the JLABS @ ... to be the first Tennessee -based ...
(Date:4/26/2017)... ... April 26, 2017 , ... Led ... phase I clinical trials comes to Tampa, San Francisco and Boston in 2017. ... representing FDA regulated organizations such as Pfizer Inc., Teva Pharmaceuticals, Advaxis, Inc., Ocular ...
(Date:4/26/2017)... ... April 26, 2017 , ... As ... conversations among healthcare industry stakeholders, the discussion surrounding the topic will continue at ... 15-18, 2017 in Los Angeles, Calif. Hosted by the Workgroup for Electronic Data ...
Breaking Biology Technology:
(Date:4/5/2017)... SEATTLE , April 5, 2017  The Allen ... the Allen Cell Explorer: a one-of-a-kind portal and dynamic ... large-scale 3D imaging data, the first application of deep ... edited human stem cell lines and a growing suite ... the platform for these and future publicly available resources ...
(Date:4/4/2017)... , April 4, 2017   EyeLock LLC , ... that the United States Patent and Trademark Office (USPTO) ... covers the linking of an iris image with a ... and represents the company,s 45 th issued patent. ... is very timely given the multi-modal biometric capabilities that ...
(Date:3/30/2017)... KONG , March 30, 2017 The ... a system for three-dimensional (3D) fingerprint identification by adopting ground breaking ... into a new realm of speed and accuracy for use in ... at an affordable cost. ... ...
Breaking Biology News(10 mins):