Flow cytofluorometric analysis of insulin binding and internalization by swiss 3T3 cells

Abstract

The binding of a fluorescein-isothiocyanate derivative of insulin to Swiss 3T3 cells was measured by flow cytometry. The kinetics of the subsequent internalization were also measured; at a concentration of 1 μM labeled insulin approximately 25% of the internalization was insulin-specific. The kinetics of endocytosis were contrasted to those of fluorescent derivatives of histone and dextran. In addition, the fusion of endocytic vesicles containing insulin or dextran with lysosomes was detected by measuring the pH-dependent increase in fluorescein fluorescence caused by the addition of chloroquine. The application of these results to the analysis of growth control by insulin and related hormones is discussed.