ISSN:
1432-0428
Keywords:
Insulin receptors
;
fluorescein-conjugated insulin
;
fluorescence activated cell sorter analysis
;
heterogeneity in insulin receptor density
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Summary 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 ∼1.0 was found to have the highest affinity to the receptor, the highest biological activity (∼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 ∼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. However, the latter method also permits selective detection of the number of high affinity insulin receptors, which cannot be done with FITC-insulin/ FACS that has a lower level for detection of fluorescence on ∼2000–3000 fluorescein molecules per cell. The FITC-insulin/FACS methodology permitted identification and isolation of viable cellular subsets within a cell population as based on the number of insulin receptors and was also used to study variations in insulin receptor density in human leucocytes. The method should make it possible to perform a number of hitherto unfeasible analyses of the biology of insulin receptor expression on eukaryotic cells.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00265023
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