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Use of FlashPlate Technology for In Vitro Measurement of [35S]-GTPγS Binding in CHO Cells Expressing the Human 5-HT1B Receptor

>The FlashPlate was then incubated for 30 minutes at 30C. To start the reaction, 50 μl [35S]-GTPγS (200 pM) was added, and the plates were shaken for 30 seconds. After a further 30 minutes, the reaction was stopped by aspirating the contents of each well. The FlashPlate was then immediately placed on a Packard TopCount Microplate Scintillation and Luminescence Counter to measure bound radioactivity.

Results

Several experiments were performed to determine the optimum conditions for binding in FlashPlate coated with CHO cell membranes expressing the h5-HT1B receptor. The maximum response produced by 5-HT [10 μM] was achieved when approximately 20 μg of membrane protein was used to coat each well and 10 μM GDP was used in the assay (Figure 1a). This is further supported by Figures 1b and 1c, which show complete 5-HT concentration response curves. The best signal-to-noise ratio was seen at the above-mentioned protein and GDP concentrations (Figures 1b and 1c, respectively). Figure 1d represents a concentration response curve for GTPγS and shows that concentrations in excess of 10 nM are adequate to estimate non-specific binding.

Intra-assay variation was investigated by measuring the stimulation of [35S]-GTPγS binding produced by 5-HT [10 μM] in 80 of the FlashPlates 96 wells. Figure 2a shows the data in terms of absolute counts per minute; Figure 2b shows the same data with each row expressed as a percentage of its individual basal value. Preliminary results on inter-assay variation over three experiments indicate only up to 6% error on mean values (Figure 3a). Further experiments would be required to provide more detail on variability. For comparative studies, concentration response curves to 5-HT, GR127935 and SB-224289 were carried out using FlashPlate and the filtration method (Figures 3a and 3b, respectively). In both as
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