5-CARBOXYFLUORESCEIN DIACETATE AS A PROBE FOR MEASURING THE GROWTH OFKERATINOCYTES

There is a requirement for a convenient and reliable method for evalua ting 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 c ompared. The methods used were the measurement of total cell protein, cell counting using an electronic Coulter counter and a fluorimetric a ssay employing 5-carboxyfluorescein diacetate (CFDA). The CFDA assay s howed the highest correlation with seeding density of the L929 cells, and the lowest standard deviations. It was the most rapid and convenie nt method for processing large numbers of samples. Only viable cells c an deacetylate the non-fluorescent CFDA to carboxyfluorescein, which i s fluorescent and accumulates inside the cells. Therefore, the assay s pecifically quantifies only viable cells. Subsequently, this assay has been successfully applied to the measurement of human keratinocyte gr owth rate on collagen gels and sponges. We have demonstrated that kera tinocytes grow equally well on gels and sponges, and that media contai ning low calcium concentrations (0.09 mM) favour rapid proliferation o f keratinocytes. Our results show that the CFDA assay is an accurate, reliable and convenient method for quantifying cell growth in vitro. I t is particularly valuable when growing cells on optically opaque subs trata, such as collagen sponges, where growth cannot be monitored dail y by microscopy.