Due C, Linnet K, Langeland Johansen N, Olsson L
Diabetologia. 1985 Oct;28(10):749-55. doi: 10.1007/BF00265023.
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)
真核细胞群体中胰岛素受体表达的多样性可用与胰岛素受体具有高亲和力的荧光染料偶联试剂结合流式细胞术进行研究。我们研究了异硫氰酸荧光素（FITC）偶联胰岛素与荧光激活细胞分选仪（FACS）联合应用以分析胰岛素受体表达的最佳条件，还研究了该方法用于识别和分离细胞群体中胰岛素受体表达存在差异的活细胞亚群的可行性。半合成人胰岛素与FITC偶联，产生了至少四种对胰岛素受体具有不同亲和力的FITC - 胰岛素分子。每种类型的FITC - 胰岛素通过半制备反相高压液相色谱法分离。发现荧光素/蛋白质比率约为1.0的制剂对受体具有最高亲和力、最高生物活性（约为天然胰岛素的50%）以及与天然胰岛素相似的抗原性。确定了关于pH、孵育时间和细胞数量的最佳染色条件，在某些方面与用125I - 胰岛素标记不同。FITC - 胰岛素与细胞的结合是饱和的，并且可以被未标记的胰岛素取代。荧光信号可通过校准曲线转换为荧光素分子的绝对数量，并且从F/P比率约为1.0计算出特异性结合的FITC - 胰岛素分子的绝对数量。FITC - 胰岛素/FACS方法允许估计胰岛素受体的总数（高亲和力和低亲和力），并且获得的数据与125I - 胰岛素结合数据的Scatchard图结果相关性良好。（摘要截短于250字）
