Resazurin-based assay for quantifying living cells during alkaline phosphatase (ALP) release

Abstract

Alkaline phosphatase (ALP) is an important reporter gene in the gene expression system, therefore monitoring cellular behavior including cell viability during ALP release is of significance. This assay produced a quantitative resazurin-based assay for cell viability in embryonic and cancer cells during alkaline phosphatase (ALP) release. A post-confluence culture method was applied to induce ALP in the cells of Balb/c 3T3, A549, MCF-7, and Ht-29. The density of each cell type was optimized using the standard cell culture assay. The main parameters affecting the results of resazurin involve the concentration of resazurin, incubation time, and cell number. The redox reaction, in which resazurin is reduced by the cells, was measured by fluorescence at 544 nm and 590 nm. The obtained data were compared with the hemocytometer assay. ALP release was determined using the optical active substrate p-nitrophenyl phosphate and colorimetric assay.