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ATLA - ISI
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Alternatives to Laboratory Animals - ATLA

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The development of a standardised protocol to measure squamous differentiation in stratified epithelia, by using the fluorescein cadaverine incorporation technique.


Gray, A.C., Malton, J. and Clothier, R.H.

ATLA, 32, 91100 (2004).

Fluorescein cadaverine (FC) incorporation into cornified envelopes during squamous differentiation in stratified epithelia acts as a fluorescent substitute for endogenous transglutaminase substrates that can be visualised and quantified. The FC incorporation technique has been used to evaluate squamous differentiation in keratinocytes cultured in a medium that stimulates differentiation and in response to modulation by chemicals. A Standard Operating Procedure for the measurement of squamous differentiation is required as part of the prevalidation procedure for in vitro assays. In the present study, keratinocytes were isolated from the epidermis of 34 human donors. Cellular metabolic activity (resorufin production), total protein (kenacid blue uptake) and transglutaminase activity (FC incorporation) were measured in 87 and 21 independent runs at 6 and 12 days, respectively. Analysis of the control data showed that the cultures had a mean resorufin produc