| Assay Method Information | |
| | FLIPR Assay |
| Description: | At the assay day cells were washed 3× with assay buffer, 10 μL buffer remained in the wells after washing. 10 μL Ca kit loading buffer (AAT Bioquest) was added to the cells and the plates were incubated with lid for 60 minutes at r.t. 20 μl assay buffer containing 60 μM glycine (20 μM final) and 3 μM glutamate (1 μM final) was added to column 1-23. Fluorescence (indicating the calcium influx as a result of the NR1/NR2B ion channel activation) was read on the FLIPRtetra device for 60 seconds to monitor the glutamate induced effects. After 2 minutes 20 μL of compound or controls (row 1-22) in assay buffer were carefully added to the wells. Fluorescence was read on the FLIPR tetra device for additional 6 minutes to monitor the compound induced effects after activation by agonists. The average of 2 measurements at 5 minutes and 5 mM 10 seconds after compound addition is calculated and further used for IC50 calculations. Each assay microtiter plate contained wells (in column 23 or 24) with DMSO controls instead of compound as controls for glycine/glutamate induced fluorescence (high controls) and wells with 1 μM of a reference NR2b NAM as low controls (Compound 22; reference: Layton, Mark E et al, ACS Chemical Neuroscience 2011, 2(7), 352-362). |
| Affinity data for this assay | |
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