| Assay Method Information | |
| | In Vitro Radioligand Binding Assay |
| Description: | The affinity (Ki) of compounds for the benzodiazepine site of human recombinant GABAARs was measured by their ability to inhibit the binding of the selective benzodiazepine antagonist [3H]Ro15-1788 ([3H] flumazenil).Mouse L(tk−) cells stably expressing human α1β3γ2, α2β3γ2, α3β3γ2, α5β3γ2 GABAARs were generated by transfection of the individual subunits in the dexamethasone-inducible expression vector pMSGneo in mouse L(tk−) cells (Hadingham et al., 1993, Mol. Pharmacol. 43:970-975 and 1993, Mol. Pharmacol. 44:1211-1218).L(tk−) cells stably expressing human α1β3γ2, α2β3γ2, α3β3γ2, α5β3γ2 GABAARs were maintained in DMEM F12 medium supplemented with 10% Foetal Bovine Serum, 1% Penicillin/Streptomycin and 1 mg/mL Geneticin G418 in an incubator at 37° C. with a humidified atmosphere with 5% CO2. Dexamethasone was added to the culture medium to induce GABAAR expression.L(tk−) cells were harvested and membranes were prepared for each receptor combination in either TE buffer (10 mM Tris·CI/0.1 mM EDTA, pH 7.5) or phosphate buffer (K2PO4 10 mM, pH 7.0) (Hadingham et al. (1992) Proc. Natl. Acad. Sci. USA; 89 (14): 6378-82) |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |