Detection of beta-adrenergic receptors on rabbit mononuclear cells isolated free of significant contamination by other cell types.
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.
D000818:Animals; D001491:Basophils / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D008214:Lymphocytes / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism*; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*
D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; C011878:iodohydroxybenzylpindolol; D010869:Pindolol