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Detection of beta-adrenergic receptors on rabbit mononuclear cells isolated free of significant contamination by other cell types
Authors:T B Casale  M Halonen  M Kaliner
Institution:1. From the Allergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205, USA;2. Division of Respiratory Sciences, University of Arizona College of Medicine, Tucson, Arizona 85724, USA
Abstract:In order to study rabbit mononuclear cell surface receptors, it was necessary to develop a procedure to isolate mononuclear cell preparations that are free of significant contamination by other cell types, especially platelets. Centrifugation of dextran-sedimented, anti-coagulated whole blood through Hypaque (density 1.060) at 600 X g for 5 min at 22 degrees C eliminated greater than 93% of starting platelets. A second 5-min Hypaque centrifugation of Hypaque-Ficoll-isolated mononuclear cells (MNC) (approximately 80% lymphocytes) at 450 X g for 5 min at 22 degrees C reduced platelet contamination to less than one platelet per three MNC, and resulted in the overall removal of greater than 99.5% of starting platelets. These relatively pure MNC which were isolated in less than 2 hr were identified as having beta-adrenergic receptors by radioligand binding techniques using 125I]iodohydroxybenzylpindolol ( 125I]IHYP). Binding of 125I]IHYP to intact rabbit MNC was a saturable, stereospecific, and rapid process with a dissociation constant (KD) of 0.53 +/- 0.18 nM and a binding capacity of 3,461 +/- 235 sites/cell.
Keywords:Address reprint requests to Thomas B  Casale  M  D    National Institutes of Health  Building 10  Room 11C207  Bethesda  Maryland 20205
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