Gf b filters

Commercial membranes of CHO cells (Perkin Elmer, Inc., Waltham, MA, USA) stably expressing human opioid receptors and the competing radioligands, [³H]DAMGO, [³H]deltorphin-2 and [³H]U-69593 for MOP, DOP and KOP, respectively, were used. Membranes were incubated in 0.5 mL of 50 mM Tris/HCl (pH = 7.4), 0.5% bovine serum albumin (BSA), with a number of peptidase inhibitors (bacitracin, bestatin, captopril) and various concentrations of radioligands for 2 h at 25 °C. Non-specific binding was assessed in the presence of 10 mM naloxone. Assays were incubated at room temperature for 1 h and reactions were terminated via filtration through Whatman GF/B filters, soaked in 0.5% polyethyleneimine (PEI), using Millipore Sampling Manifold (Billerica, MA, USA). Radioactivity was determined after an 8 h extraction period [69 (link)].

The binding of [N-methyl-³H]scopolamine ([³H]NMS) was done as previously described
[17 ]. Briefly, ASMCs were serum deprived for 24 h and then incubated for 3, 7 or 15 days with 10% FBS, ACh (10^-5 M) or CCh (3 × 10^-7 M). Control cells were incubated in serum free medium. Cells were lysed by scraping in buffer (25 mM Tris-HCl, 2.5 mM CaCl₂, 1 mM PMSF, 1 mM Pefabloc and 10 μg/ml aprotinin). Cell extracts were homogenized with 5 strokes in a Heidolph Silent Crusher S (Heidolph Instruments GmbH & Co. KG, Germany) and the homogenate was centrifuged at 10000 g for 5 min at 4°C. The supernatant was diluted up to 11 ml in assay buffer (10 mM HEPES, 100 mM NaCl, 10 mM MgCl₂) and centrifuged at 150000 g for 30 min at 4°C. The pellet was then resuspended in assay buffer and subjected to sonication for 5 sec. 30 μg of protein was added in the reaction, along with 5 nM of [³H]NMS. Nonspecific binding was estimated by the presence of 100 μM atropine. After 80 min of incubation in order to allow radioligand binding only to M₃ receptors
[17 ], the samples were filtered through GF/B filters (Millipore) using a Millipore vacuum filtration system (EMD Millipore Corporation, Billerica, MA, USA). Before radiation counting in a Wallac β-counter, filters were dried overnight and incubated in Ultimate gold XR (Perkin Elmer) scintillation fluid for 1 h at RT.