5-Carboxyfluorescein Diacetate as a Probe for Measuring the Growth of Keratinocytes

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Abstract
There is a requirement for a convenient and reliable method for evaluating the growth rate of human keratinocytes cultured on collagen-based substrates. Therefore, three methods of determining cell growth were first used to quantify the growth rate of the well-characterised L929 mouse fibroblast cell line on tissue culture plastic and the results compared. The methods used were the measurement of total cell protein, cell counting using an electronic Coulter counter and a fluorimetric assay employing 5-carboxyfluorescein diacetate (CFDA). The CFDA assay showed the highest correlation with seeding density of the L929 cells, and the lowest standard deviations. It was the most rapid and convenient method for processing large numbers of samples. Only viable cells can deacetylate the non-fluorescent CFDA to carboxyfluorescein, which is fluorescent and accumulates inside the cells. Therefore, the assay specifically quantifies only viable cells. Subsequently, this assay has been successfully applied to the measurement of human keratinocyte growth rate on collagen gels and sponges. We have demonstrated that keratinocytes grow equally well on gels and sponges, and that media containing low calcium concentrations (0.09 mM) favour rapid proliferation of keratinocytes. Our results show that the CFDA assay is an accurate, reliable and convenient method for quantifying cell growth in vitro. It is particularly valuable when growing cells on optically opaque substrata, such as collagen sponges, where growth cannot be monitored daily by microscopy.