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
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.
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