Relationship between cell differentiation and binding of fluorescein isothiocyanate (FITC)-conjugated insulin of keratinocytes in culture.

Basal-type keratinocytes, isolated from newborn rat skin and separated on Percoll density gradients, proliferate in low (0.1 mM) calcium medium and, after raising the calcium level to normal (1.96 mM), stratify. Cells in the low calcium culture do not have extensive cell-cell connections, as seen with fluorescein isothiocyanate (FITC)-conjugated insulin. Fluorescein isothiocyanate-conjugated concanavalin A and Griffonia simplicifolia isolectin B4, but not peanut agglutinin (PNA), fluorescently label these cells. In 3-day-old low calcium cultures, within 2 h after raising the calcium of the medium to the normal level, intense binding of PNA to cells appears and neighboring cells are connected through bundles of filaments that are fluorescently labeled by FITC-insulin. After 2 days in normal calcium medium, the cultures exhibit relatively smooth, straightlined, cell boundaries that are labeled by FITC-insulin and cell boundaries and intracellular granules that are stained by hematoxylin. One day later, similar cell boundaries are present, but they are not significantly decorated by FITC-insulin and, under phase contrast microscopy, are dark. Free FITC gives labeling patterns similar to those given by FITC-insulin, but the FITC labeling is blocked by mercaptoethanol and dithiothreitol in contrast to FITC-insulin binding. The present results suggest the insulin moiety is involved in the labeling by FITC-insulin and the labeling is chronologically related to the stage of cell differentiation.