Assay Method Information

Assay Name:  Radioligand Binding Assays
Description:  The composition of the assay buffers was as follows: for 5-HT1AR: 50 mM TrisHCl, 0.1 mM EDTA, 4 mM MgCl2, 10 lM pargyline, and 0.1% ascorbate; for 5-HT6R: 50 mM TrisHCl, 0.5 mM EDTA, and 4 mM MgCl2; and for 5-HT7bR: 50 mM TrisHCl, 4 mM MgCl2, 10 lM pargyline, and 0.1% ascorbate. All assays were incubated in a total volume of 200 L in 96-well microtiter plates for 1 h at 37 C, except for 5-HT1AR which were incubated at room temperature for 1 h. The process of equilibration is terminated by rapid filtration through Unifilter plates with a 96-well cell harvester, and radioactivity retained on the filters was quantified on a Microbeta plate reader. For displacement studies, the assay samples contained as radioligands: 1.5 nM [3H]8-OH-DPAT (187 Ci/mM) for 5-HT1AR; 2 nM[3H]LSD (85.2 Ci/mM for 5-HT6R or 0.6 nM [3H]5-CT (39.2 Ci/mM) for 5-HT7R. Non-specific binding was defined with 10 M of 5-HT in 5-HT1AR and 5-HT7R binding experiments, whereas 10 M methiothepin was used in 5-HT6R assa
Affinity data for this assay

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