| Assay Method Information | |
| | HTRF cAMP Assay |
| Description: | Compounds of the present invention were evaluated using the human H3 receptor (H3R) HTRF cAMP assay. In this assay, HEK293 cells expressing the human H3 receptor were suspended in PBS containing 100 μM IBMX and plated into 384-well assay plates (Perkin Elmer Proxiplate 384-Plus; 15,000 cells per well; 5 μL plating volume) and allowed to equilibrate for an hour. Test compounds were serially diluted in 100% DMSO and then further diluted in PBS containing forskolin (2 μM). Test compounds (5 μL) were then added to the assay plate and the mixture was incubated for 1 hour. HTRF assay reagents (Cisbio, Dynamic 2 cAMP Kit), cAMP-d2 and cryptate-labeled anti-cAMP antibody, are mixed with cell lysis buffer and added to the assay plate. After 1-hour incubation with these reagents, the assay plate was read on an HTRF-compatible microplate reader (Perkin Elmer EnVision or BMG Pherastar). |
| Affinity data for this assay | |
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