Assay Report Card

ID: ALA3888188

Type: Binding

Description: I1 Receptor Binding Assay: Binding assays were performed at 37° C. using [125I]LNP 911 as radioligand following the general procedure described but adapted to washed whole platelets (Greney, H.; Urosevic, D.; Schann, S.; Dupuy, L.; Bruban, V.; Ehrhardt, J,-D.; Bousquet, P.; Dontenwill, M. [125I]2-(2-Chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP911), a High-Affinity Radioligand Selective for I1 Imidazoline Receptors. Mol. Pharmacol. 2002, 62, 181-191).Incubation was initiated through the addition of 900 to 950 ul of platelet suspension at a concentration of 500000/ul in a final volume of 1 ml Tyrode albumin and was performed at 37° C. for 5 min (equilibrium conditions). The competitive assays were conducted using a single concentration of radioligand (50 uM, 200 000 cpm), in the presence of increasing concentrations of suitable non-labelled ligand. Non-specific binding was determined by the binding of [125I]LNP 911 in the presence of 100 nM of non-labelled LNP 911 and represented about 10% of total radioactivity when 50 uM of [125I]LNP 911 were used. The reaction was halted by rapid vacuum filtration through GF/C fibre glass filters followed by five rapid washings of the filters with 3 ml of ice-cold Tyrode (137 nM NaCl, 2.7 nM KCl, 12 nM NaHCO3, 0.36 nM NaH2PO4, pH 7.35). Radioactivity was measured using a gamma counter (Wallac 1410).