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| Solution Information | help | |
| Enzyme: | Metabotropic glutamate receptor 1 | |
| inhibitor: | BDBM55081 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Basic protocol: 1. Cells are plated at a density of 20,000 cells/20 ul well 1 day prior to assay in Assay Media (DMEM with 20mM HEPES, 10% dialyzed FBS, 1mM sodium pyruvate). 2. The following day, media is removed from the cells. 3. 20 ul/well of Fluo-4 AM dye (1 uM) in Assay Buffer (HBSS + 2.5mM probenecid) is added. 4. The plates are incubated for 45 min to one hour at RT. 5. Dye is removed from the cells. 6. 20 ul of assay buffer is added and removed. 20 ul of assay buffer is added. 8. Plates are loaded into a Hamamatsu FDSS. 9. Compounds are tested for selectivity at a 10 uM final concentration (prepared as a 2X stock, 20 ul added per well) and the agonist is added at a 5X concentration (10 ul of agonist added per well, 50 uL total volume). A baseline read is taken for 1.5 seconds, the first addition (compound or DMSO-matched vehicle) occurs at 1.5 seconds and the second addition (agonist) is added at 142 seconds. The read continues for 300 seconds. Data analysis: Data were analy | |
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