| Assay Method Information | |
| | Human D1 Receptor PAM Assay |
| Description: | Test compound is serially diluted (1:2) with DMSO into assay plates (ProxiPlate-384 Plus, PerkinElmer) using acoustic dispensing (Labcyte) to provide 20 concentrations for full response curves. Test compound (80 nL) is added to 5 μL STIM buffer containing 2000 cells, and 5 μL of a 2 concentration dopamine solution in STIM buffer that will generate an EC20 level response (24 nM in stock solution, or 12 nM final) and a final DMSO concentration in the well of 0.8%. Plates are incubated at room temperature for a total reaction time of 60 min. cAMP production is quantified using HTRF detection (Cisbio) according to the manufacturer's instructions. Generally, lysis buffer containing anti-cAMP cryptate (5 μL) and D2-conjugate (from HTRF kit)(5 μL) is added to the wells, plates are incubated for an additional 60-90 min, and the time-resolved fluorescence is detected using an EnVision plate reader (PerkinElmer). Fluorescence data is converted to cAMP concentrations using a cAMP standard curve and analyzing using a 4-parameter non-linear logistic equation (Genedata Screener, version 13.0.5-standard). |
| Affinity data for this assay | |
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