Fluorescein cadaverine incorporation as a novel technique for the characterization of terminal differentiation in keratinocytes

A novel technique for detecting transglutaminase activity and the productio n of cornified envelopes in keratinocytes has been devised. This was based on the enzymatic incorporation of fluorescein-labelled cadaverine (FC) into cornified envelopes. The addition of FC (20 mu m) to the incubation medium served as an amine donor for transglutaminase reactions in place of protei n lysine residues. Cells incorporating the label became visible with fluore scence microscopy and were quantified by fluorimetry. There was a significa nt difference in the level of FC incorporation into cornified envelopes und er the various media conditions and time points employed. The greatest inco rporation was observed by keratinocytes cultured in Green's medium and fluo rescent intensity decreased in the order: Green's > KGM with calcium > KGM. Confocal imaging of keratinocytes dual stained with FC and propidium iodid e revealed the presence of distinct layers and demonstrated how FC was inco rporated into differentiating cells and not the basal layer. FC incorporati on has the potential to serve as a rapid assessment of terminal differentia tion in keratinocytes. It is simple and less time-consuming than currently available alternative techniques. This approach also has the advantage of c ombining microscopic and quantitative data. (C) 1999 Elsevier Science Ltd. All rights reserved.