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2, and 0.1% (w/ v) bovine serum albumin, and the total volume was 0.5 mL. Nonspecific was defined with 0.1 mM YM-09151. Following incubation, samples were filtered using a Canberra Packard Filtermate and washed four times with ice-cold 50 mM Tris (pH 7.4 at 37°C). The radioactivity on the filters was measured using a Canberra Packard Topcount. Competition curves were analysed using INFLEXION (Bowen, W. P.; Jerman, J. C. Inflexion: Automated analysis of radio-ligand binding data with Microsoft Excel. Br J. Pharmacol. 1994, 113, 440P).
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RoboFit: A Versatile Macro-Driven Template for Curve Fitting, Analysis and Presentation in Microsoft Excel
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2, before changing to medium without FCS. After a further 16-18 h, cups were loaded into the sensor chambers of the microphysiometer and the chambers perfused with running medium (bicarbonate-free Dulbecco's modified Eagles medium containing 2 mM glutamine and 44 mM NaCl). For agonist experiments, cells were exposed to increasing concentrations of agonist at half-hour intervals. For antagonist experiments, cells were exposed five times (at half-hour intervals) to a single concentration of quinpirole (30 nM) before addition of the first antagonist concentration. After a 30 min interval, cells were again stimulated with quinpirole (in the continued presence of the antagonist), before the second (higher) antagonist concentration was applied. In all, responses in the presence of five increasing concentrations of antagonist were determined. Peak acidification rate to each agonist concentration was determined and concentration-response curves fitted using RoboFit (Tilford N. S.; Bowen, W. P.; Baxter, G. S. RoboFit: A Versatile Macro-Driven Template for Curve Fitting, Analysis and Presentation in Microsoft Excel. Br. J. Pharmacol. 1995, 115, 160P).
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