Analysis of insulin receptors on heterogeneous eukaryotic cell populations with fluorochrome-conjugated insulin and fluorescence-activated cell sorter. Advantages and limitations to the 125I-labelled insulin methodology.
The diversity in insulin receptor expression within eukaryotic cell populations can be studied with fluorochrome conjugated reagents with high affinity to the insulin receptor in combination with flow cytometry. We studied the optimal conditions for application of fluorescein isothiocyanate (FITC) conjugated insulin in combination with the fluorescence activated cell sorter (FACS) to analyse insulin receptor expression, and also studied the feasibility of this method for identifying and isolating viable subsets with differences in insulin receptor expression within a cell population. Semisynthetic human insulin was conjugated to FITC, which resulted in at least four types of FITC-insulin molecules with different affinities to the insulin receptor. Each type of FITC-insulin was isolated by semipreparative reverse phase high pressure liquid chromatography. The preparation with a fluorescein/protein ratio of approximately 1.0 was found to have the highest affinity to the receptor, the highest biological activity (approximately 50% of native insulin), and similar antigenicity as native insulin. The optimal staining conditions with respect to pH, time of incubation, and cell number were determined, and were different in some aspects from labelling with 125I-insulin. The binding of FITC-insulin to cells was saturable and could be displaced with unlabelled insulin. The fluorescence signal could be converted to absolute numbers of fluorescein molecules by a calibration curve, and the absolute number of specifically bound FITC-insulin molecules calculated from a F/P ratio approximately 1.0. The FITC-insulin/FACS method permits estimation of the total number of insulin receptors (high plus low affinity), and the data obtained correlate well with the results from Scatchard plot of 125I-insulin binding data.(ABSTRACT TRUNCATED AT 250 WORDS)
Due
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Linnet
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Langeland Johansen
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Olsson
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D000328:Adult; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D002469:Cell Separation; D000939:Epitopes / Q000276:immunology; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008214:Lymphocytes / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D013861:Thiocyanates
D000906:Antibodies; D000939:Epitopes; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007328:Insulin; D007457:Iodine Radioisotopes; D013861:Thiocyanates; C031573:insulin, fluorescein-isothiocyanated-; D011972:Receptor, Insulin; D016650:Fluorescein-5-isothiocyanate