| Assay Method Information | |
| | HTRF-Based IP1 Accumulation Assay |
| Description: | The 5-HT2A receptor antagonistic effect of the test compounds was confirmed by measuring the production amount of IP1 using the HEK293 cell line permanently overexpressing the human 5-HT2A receptor. The test compounds were dissolved in 100% DMSO to a final concentration of 10 mM. The starting concentration of 10 μM was prepared from the stock solution, followed by serial dilutions to prepare working solutions in a buffer containing lithium chloride (LiCl). The cells and compounds prepared in the test buffer were placed in a 96-well plate and incubated at room temperature for 10 minutes. Serotonin of EC80 concentration was added, followed by incubation at 37° C. for 30 minutes in a light-shielding state. To measure IP1 accumulation in cells, IP1-d2 acceptor and IP1-Cryptate donor—which are HTRF-based substances for fluorescence signals—were added and incubated for 1 hour at room temperature in a light-shielded state. The fluorescent emission signals of acceptor and donor were detected, and HTRF ratio values were substracted by mean value of serotonin control. |
| Affinity data for this assay | |
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