Réaction #73866

Équation de réaction

Tris-HCl

[3H]-ketanserin

DMSO

[3H]-ketanserin

→

Serotonin

Conditions de réaction

Traitement

1. 1
   Autrefor 15 minutes
2. 2
   Autreat 37° C.
3. 3
   workup.ADDITIONit was quickly added on a glass fiber
4. 4
   Filtrationfilter plate (Multiscreen FB, Millipore inc.) coated with 0.05% Brij 35
5. 5
   Filtrationfiltered under reduced pressure
6. 6
   FiltrationThe glass fiber filter
7. 7
   Lavagewas washed with 200 μL of ice-
8. 8
   Températurecooled 50 mM Tris-HCl (pH=7.6) twice
9. 9
   Filtrationrepeatedly filtered under reduced pressure
10. 10
    workup.ADDITIONa vial containing 4 mL of Ecoscint A (National Diagnostics inc.)
11. 11
    FiltrationThe residual radioactivity on the glass fiber filter

Mode opératoire

5-HT2A receptor binding assay was performed in reference to a method described by Hirose et al. (Japan. J. Pharmacol., 53, 321-329, 1990). After a reaction in a total volume of 200 μL of 50 mM Tris-HCl (pH=7.6) buffer solution containing 50 μL of [3H]-ketanserin (final concentration 1 nM), 1 μL of test drug in DMSO, and 149 μL of human 5-HT2A receptor-expressing CHO cell membrane sample, human 5-HT2A receptor binding activity of [3H]-ketanserin was measured. The reaction solution let stand for 15 minutes at 37° C., and then it was quickly added on a glass fiber filter plate (Multiscreen FB, Millipore inc.) coated with 0.05% Brij 35 and filtered under reduced pressure. The glass fiber filter was washed with 200 μL of ice-cooled 50 mM Tris-HCl (pH=7.6) twice, repeatedly filtered under reduced pressure, and then transferred to a vial containing 4 mL of Ecoscint A (National Diagnostics inc.). The residual radioactivity on the glass fiber filter was measured by a liquid scintillation counter. Nonspecific binding was measured in the presence of 10 μM of MDL-100907, and [3H]-ketanserin binding inhibition rate in the presence of 1 nM or 10 nM of test drug was determined. The results are shown in Tables 8 to 13.

Source