Assay-based quantitative analysis of PC12 cell differentiation.

Differentiation of PC12 cells has been quantified by measurement of neurite length. However, this procedure is not suitable for large numbers of samples, for example in 96-well tissue culture plates. For this reason, we established three simple and quantitative methods for nerve growth factor-induced differentiation of PC12 cells cultured in 96-well plates. Firstly, because neuronal markers, including neurofilament proteins and β-tubulin isotype III, are increased during PC12 cell differentiation, we developed cell enzyme-linked immunoabsorbent assays (ELISA)-based procedures that measure the amount of these proteins. Secondly, because lactate dehydrogenase (LDH) is down-regulated and mitochondrial NADH-dehydrogenase activity is increased during PC12 cell differentiation, we established procedures to measure changes in LDH and NADH dehydrogenase. We found that the cell ELISA and cell counting assays could be used to determine the degree of PC12 cell differentiation caused by nerve growth factor, basic fibroblast growth factor and epidermal growth factor. However, neither LDH nor NADH-dehydrogenase activities changed during Thy-1 antibody-induced differentiation. These findings show that in addition to the cell ELISA procedures, the LDH and NADH-dehydrogenase procedures are useful for characterization of growth factor-induced PC12 cell differentiation.