Fluorescein cadaverine incorporation as a novel technique for the characterization of terminal differentiation in keratinocytes.
A novel technique for detecting transglutaminase activity and the production 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 (20mum) to the incubation medium served as an amine donor for transglutaminase reactions in place of protein lysine residues. Cells incorporating the label became visible with fluorescence microscopy and were quantified by fluorimetry. There was a significant difference in the level of FC incorporation into cornified envelopes under the various media conditions and time points employed. The greatest incorporation was observed by keratinocytes cultured in Green's medium and fluorescent intensity decreased in the order: Green's> KGM with calcium>KGM. Confocal imaging of keratinocytes dual stained with FC and propidium iodide revealed the presence of distinct layers and demonstrated how FC was incorporated into differentiating cells and not the basal layer. FC incorporation has the potential to serve as a rapid assessment of terminal differentiation in keratinocytes. It is simple and less time-consuming than currently available alternative techniques. This approach also has the advantage of combining microscopic and quantitative data.
Gray
A C
AC
FRAME Alternatives Laboratory, School of Biomedical Sciences, Medical School, University of Nottingham NG7 2UH, UK.
Garle
M J
MJ
Clothier
R H
RH