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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# 655
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