| Assay Method Information | |
| | FLIPR Assay |
| Description: | Activity of compounds on native GABAA receptors is evaluated by monitoring calcium fluxes using a 96 well format FLIPR system (Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices). Briefly, cortical embryonic neurons are dissociated from E18 rat embryos and plated at optimum density into black-walled, transparent bottom poly-D-lysine coated 96-well FLIPR plates. After loading the cells with a calcium sensitive dye (Fluo4-AM, Molecular Devices), the cells are bathed in a solution containing low chloride (chloride replaced by gluconate). Under these conditions activation of GABAA receptors causes an efflux of chloride ions (in the direction of the chemical gradient), which results in membrane depolarization and consequently activation of voltage gated calcium channels (VGCCs). Calcium influx through VGCCs is recorded and analysed offline using the FLIPR system. For a pharmacological validation of the assay, concentration response curves (CRC) are recorded for the standard agonist (GABA) and standard antagonist (Gabazine). Any effects are determined in CRC mode against a fixed concentration of agonist GABA at 10 uM (equivalent to an EC90 GABA response). |
| Affinity data for this assay | |
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