Multiscreen fc filter type b

Isolated cell membranes were resuspended in ligand binding buffer (50 mM Tris-HCl, 10 mM MgCl2 and 1 mM EDTA, pH, 7.4) and 5 μg membrane protein/well were loaded onto poly(ethyleneimine) (0.1% v/v) treated 96-well glass fiber filter plates 49 (MultiScreen-FC filter type B, Millipore, Billerica, MA) as previously described [38 (link)]. Cells were incubated with 1.25–40 nM [³H]ZM241385 in the presence or absence of unlabeled competitor ligand (50 μM NECA) or 1.25-250 nM [³H] CGS21680 in the presence or absence of unlabeled competitor ligand (50 μM CGS21680) for 1.5 h. Once binding equilibrium was reached, membranes were washed three times with ice-cold binding buffer and then 30 μL of scintillation solution (ULTIMA gold, Perkin Elmer) was added to each well. Radioactive counts (CPMs) using a Perkin-Elmer 1450 Microbeta liquid scintillation counter were measured to determine ligand binding approximately 24 h after the addition of the scintillation solution. Non-specific binding was determined from binding to membranes in the presence of an unlabeled competitor and CPMs were subtracted from total binding to calculate specific binding.