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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# 655090). Cells were incubated either for one hour with a buffer containing 4.2 M Fluo-3 or for one-half hour with 1X Loading Buffer from the FLEXstation Calcium Assay Kit (Molecular Devices, cat#R8041) before using FLEXstation to dispense carbachol and monitor the mobilization of calcium into the cell cytosol. All experiments were performed at a final concentration of 2.5 mM probenecid in order to inhibit endogenous efflux pumps present in M1 CHO cells.
DETERMINATION OF EC50 VALUES
Cells were exposed to a range of carbachol (0 to
1 M) using half log dilutions of the agonist.
Results are representative of at least four
independent experiments performed over a
period of at least two different days. The raw
data was analyzed as Max-Min and Absolute
RFU values (Figures 1, 2). The average value of
replicate samples was plotted on a graph using a
curve fit of four parameter, for which the C
value is the concentration of drug that causes
50% of maximal response (EC50 value, see
Figure 3). The EC50 values for carbachol using
Fluo-3 ranged from 1939 nM, whereas it
ranged from 1230 nM using the FLEXstation
Calcium Assay Kit (see Figures 3 and 4).
DETERMINATION OF Z FACTOR
Cells in half of the microplate were treated with
0.5 M carbachol, while cells in the other half of
the plate were treated with 1X Reagent Buffer
(1X HBSS/20 mM HEPES pH 7.4). The
FLEXstation Calcium Assay Kit was used to
monitor carbachol-mediated calcium
mobilization (Figure 5). Z factor was calculated
using the formula2:
The terms sc- and sc+ denote the standard deviation of the negative control and positive control, respectively, whereas |c+ c-| denotes the absolute value of the difference in the mean of the positive control and the mean of the negative control. The Z factor obtained for FLEXstation using the FLEXstation Calcium Assay Kit was 0.65. This indicates a large separation band between the negative and positive controls and an optimized, high quality assay.
SUMMARY
FLEXstation generates highly reproducible
results for calcium mobilization assays used to
monitor the activity of Gq-coupled receptors
(Fig. 3). First, the EC50 values obtained for carbachol were consistent between detection
methods, Fluo-3 and the FLEXstation Calcium
Assay Kit. Also, assays analyzed on the same day
showed less than two-fold differences, and assays
analyzed on different days showed less than fourfold
differences, which further demonstrates the
reliable performance of FLEXstation (data not
shown). Lastly, the Z factor determination
indicates that FLEXstation provides an excellent
tool for assay development for the model system
examined. In addition, other benefits include
SOFTmax PRO software that makes it simple to
set up the instrument and analyze the data.
Moreover, the dual monochromators provide
increased flexibility for assay development or
basic research purposes. In conclusion,
FLEXstation provides an easy-to-use, versatile
tool to help basic research and assay
development laboratories reach their goals.
For more detailed information, please refer to
the application note Comparison of FLIPR
and FLEXstation for Calcium Mobilization
Assays, available for download from Molecular
Devices web site at:
www.moleculardevices.com/pages/flex_lit1.html
REFERENCES
1. Buck, M. A., and C. M. Fraser. 1990. Biochem. Biophys. Res. Commun. 173: 666672.
2. Zhang, J-H., T.D.Y. Thomas, and K. R. Oldenburg. 1999. J. Biomol. Screening 4: 6773.
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