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Gq-coupled receptor assays: a comparison of FLEXstation and FLIPR

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
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