Assay Method Information

Assay Name:  5-HT3 Receptor Assay
Description:  Membranes from HEK293 cells which express recombinant human 5-HT3 receptor (RB-HS3, Receptor Biology, Inc., MD, USA) are diluted in accordance with the manufacturer's instructions in incubation buffer (50 mM tris base, pH 7.4, 5 mM MgCl2, 0.5 mM EDTA, 0.1% (w/v) ascorbic acid, 10 μM pargyline and incubated in a volume of 200 μl (amount of membrane protein 3 μg) in the presence of 0.5 nM of the selective 5-HT3 receptor radio ligand [3H]-GR65630 (NET 1011, Du Pont) and various concentrations of the test substance at 21 C. for 60 min. The nonspecific binding is determined by incubation in the presence of 100 μM 5-HT (5-hydroxytryptamine). The incubation is stopped by filtering through type A/E glass fiber filters (Gelman Sciences) or GF/B filters (Whatman) which have previously been placed in 0.3% (v/v) polyethyleneimine (PEI) for at least 1 h. The filters are washed three times with 3 ml of washing buffer (50 mM Tris-HCl, pH 7.4; 4 C.), and the bound radioactivity is determined by scintillation measurement. All the assays are carried out in triplicate. The dissociation constant Ki of the test substance is determined from the IC50 of the compounds (concentration of the test substance at which 50% of the ligand bound to the receptor is displaced), the dissociation constant KD and the concentration L of [3H]GR65630 (Ki=IC50/(1+L/KD)).
Affinity data for this assay

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