Assessment of adhesion assays for use with keratinocytes.
Cell attachment, as a biological process, is an important aspect with respect to graft survival and "take". With the ever-increasing use of cultured epithelial autografts for coverage and re-epithelialization of wounds, the assessment of keratinocyte adhesion in vitro has become a more common requirement in studies involving extracellular matrix proteins and their receptor molecules. Cell adhesion has been well-documented in immunological research, however keratinocyte adhesion has been investigated by manual counting (using methylene blue) or other less sensitive colorimetric methods. With the increase in number of fluorogenic probes available, their use as a sensitive alternative to radioactive labelling has been promoted in the literature. This study was carried out to investigate the possibility of using fluorescent probe 5,6-carboxyfluorescein diacetate succidimyl ester to achieve a more standardized assay in the assessment of keratinocyte adhesion. Adhesion was assessed on extracellular matrix proteins such as fibronectin, collagen types I & IV and laminin. We concluded that the fluorescent probe might provide greater sensitivity in measuring adhesion, however it may be cytotoxic to keratinocytes. Pre-labelling of keratinocytes may affect cellular functions such as adhesion and even proliferation and consequently the probe must be chosen with care.
May
A L
AL
The University of Western Australia, Perth.
Wood
F M
FM
Stoner
M L
ML
D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D007797:Laminin / Q000378:metabolism*; D013388:Succinimides; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005353:Fibronectins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007797:Laminin; D013388:Succinimides; D003094:Collagen; D001619:beta-N-Acetylhexosaminidases