( e Set Up The assay paramet...)Gq-coupled Receptor Assays Using FLEXstation

1. Wavelengths: The samples were excited at 485 nm, and emission was detected at 525 nm with a 515 nm cutoff filter.

2. Sensitivity: The number of reads/well was set to 6, and PMT was set to high.

3. Timing: Samples were read at 1.52 second intervals for 120 seconds (total of 79 reads/well).

4. Autocalibrate: On

5. Compound transfer: The pipettor height was set to 220 L to transfer a volume of 50 L at a rate of 4 (corresponds to a rate of 64 L/second). Compound transfer was started after 16 seconds of background reading.

RESULTS
The average EC₅₀ value obtained with the FLEXstation Calcium Assay Kit and the FLEXstation was 47 mM (Figs. 3, 4). The values varied by less than twofold for experiments performed on the same day and by less than fourfold for experiments performed on different days. The range of values from all experiments was between 24 and 87 mM. The coefficient of variation of replicate samples within an experiment varied from 5.6 to 13.8% (average 8.6%) for the 1 mM ADP concentration.

Furthermore, the EC₅₀ value obtained with the fluorescence-based whole cell method (47 M) compared favorably to the EC₅₀ value obtained by measuring [³H]IP (~190 M). Thus, the assay is highly reproducible within and between experiments as well as between assay types (IP and calcium accumulation).

CONCLUSIONS
FLEXstation can be used to assess the specificity and sensitivity of G_q-coupled receptors for different ligands and drugs that are meant to target these receptors. Specifically, FLEXstation and SOFTmax PRO software provide easy experimental set-up, gathering of data
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