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

Abstract

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 FITCinsulin 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 FITCinsulin 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-insu-Iin/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.

Inactivation of Cytochrome P450 2B1 by Benzyl Isothiocyanate, a Chemopreventative Agent from Cruciferous Vegetables

“…One could therefore assume that the reaction of BITC with exposed surface amino acid residues was not involved in substrate catalysis. Although it has been shown that the modification of rabbit P450 2B4 by fluorescein isothiocyanate caused a 25% loss in benzphetamine N-demethylation activity due to modification of the ε-amino group of lysine-382 (), the result described in this study is similar to other reports where enzyme activity was not affected even though enzyme modification clearly occurred ( , ).…”

Overcoming the Diffusion Barrier of Mucus and Absorption Barrier of Epithelium by Self-Assembled Nanoparticles for Oral Delivery of Insulin

“…F-insulin was synthesized according to the previous reported procedure. 38 The nanoparticles were characterized for particle size and zeta potential with a Malvern Zetasize NanoZS90 (Malvern Instruments Ltd., U.K.). The morphology of NCs/NPs was examined by transmission electron microscope (TEM, H-600, Hitachi, Japan).…”

“…Furthermore, fluorescent-labeled nanoparticles were prepared with the same method using FITC-labeled insulin (F-insulin). F-insulin was synthesized according to the previous reported procedure …”

Penetratin Derivative-Based Nanocomplexes for Enhanced Intestinal Insulin Delivery

“…Fluorescent labeled nanocomplexes were also prepared with the same method using FITC-labeled insulin (F-insulin). F-insulin was synthesized according to the previous report …”

“…F-insulin was synthesized according to the previous report. 17 The prepared nanocomplexes were characterized for particle size and zeta potential with a Malvern Zetasize NanoZS90 (Malvern Instruments Ltd., UK). All measurements were performed in triplicate.…”