| Assay Method Information | |
| | Radiolabel Binding Assay |
| Description: | A solution of the compound of the disclosure to be tested is prepared as a 1-mg/ml stock in Assay Buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, pH 7.4) or DMSO according to its solubility. A 1-mg/ml stock of the reference compound 5-hydroxytryptamine (5-HT) is also prepared as a positive control. Eleven dilutions (5x assay concentration) of the compound of the disclosure and 5-HT are prepared in the Assay Buffer by serial dilution to yield final corresponding assay concentrations ranging from 10 uM to 10 uM.A stock concentration of 5 nM [3H]LSD (lysergic acid diethyl amide) is prepared in Assay Buffer. Aliquots (50 ul) of radioligand are dispensed into the wells of a 96-well plate containing 100 ul of Assay Buffer. Duplicate 50-ul aliquots of the compound of the disclosure test and 5-HT positive control reference compound serial dilutions are added. Membrane fractions of cells expressing recombinant 5HT2B receptors (50 uL) are dispensed into each well. The membranes are prepared from stably transfected c ell lines expressing 5HT2B receptors cultured on 10-cm plates by harvesting PBS-rinsed monolayers, resuspending and lysing the monolayers in chilled, hypotonic 50 mM Tris-HCl, pH 7.4, centrifuging at 20,000xg, decanting the supernatant and storing at -80° C. The membrane preparations are resuspended in 3 ml of chilled Assay Buffer and homogenized by several passages through a 26 gauge needle before use in the assay. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |