Radioligand binding assay

To measure the equilibrium binding of [15-³H]-iloprost (20 Ci/mmol; American Radiolabeled Chemicals, Inc.) to IP receptor, COS-1 cells were seeded in 24-well plates. Twenty-four hours later, they were transfected with a plasmid containing human IP receptor cDNA using Fugene 6 (Promega) transfection reagent, according to the manufacturer’s instructions. Two days later, cells were rinsed once with ice-cold binding buffer (PBS + 0.5% fatty-acid free BSA) and competition binding assays were carried out by incubating transfected cells in binding buffer containing 10 nM [³H]-iloprost and indicated concentrations of unlabeled substances for 90 min at +4°C. Binding was stopped by three washing steps with ice-cold binding buffer. Thereafter, cells were lysed in lysis buffer (0.1% Triton X-200, 2N NaOH) and transferred to vials containing scintillation fluid (Ultima-Gold; Perkin-Elmer). Radioactivity was measured by a scintillation counter (Hidex 300SL).