[Quantitative assay of p-nitrophenol-N-acetyl-beta-D-glucosaminide in human keratinocyte culture]

Attachment and growth of human epidermal keratinocyte culture can be assayed by counting cells or measuring incorporation of radioactive nucleotides (3H-TdR) during cell proliferation. In this study a rapid colorimetric assay for human epidermal keratinocyte growth and viability has been developed based on the colour reaction of NAG (p-nitrophenol-N-acetyl-beta-D-glucosaminide). The results can be read on a scanning multiwell spectrophotometer and show a high degree of precision. The data of experiments show that the absorbance (OD) is directly proportional to the number of cells. 10(3)-10(6) cells per well (1.5 cm2) can be assayed by controlling the time of colour reaction. This method was used to measure keratinocyte proliferation in different culture systems of different culture conditions, growth factor and keratinocyte growth-promoting activity stimulations. The results were supported by counting cells, measuring of 3H-TdR incorporation, or analysing the area of keratinocyte confluents stained with Rhodanile blue. The main advantages of the colorimetric assay are its rapidity and precision, the avoidance of any radioisotope, and it is capable of handling large numbers of culture.