An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells

作者：Jendresen Charlotte; Daws Michael R; Nilsson Lars N G^*

来源：Journal of Pharmacological and Toxicological Methods, 2018, 90: 67-75.

DOI：10.1016/j.vascn.2017.11.004

• Methods
• Reporter cells
• Receptor stimulation
• BWZ.36
• beta-galactosidase
• Chlorophenol-red-beta-D-galactopyranoside
• CPRG

Introduction: Reporter cells expressing a chimeric receptor that activates a reporter can be used for screening ligand-mediated signal transduction. In this study, we used reporter cells harboring an NFAT/lacZ construct that express beta-galactosidase when the chimeric receptor is stimulated. A colorimetric beta-galactosidase substrate, chlorophenol-red beta-D-galactopyranoside (CPRG), was used to detect enzymatic activity. Sub-optimal conditions have unfortunately extensively been reported with such reporter-based beta-galactosidase assays. Here, we aimed to improve the CPRG-based colorimetric assay such that receptor ligands could be effectively screened with reporter cells.
Methods: After stimulation of reporter cells, we determined beta-galactosidase activity by absorbance measurement of beta-galactosidase-dependent CPRG hydrolysis. We systematically examined each component in a standard lysis buffer most commonly reported for this type of reporter cells. Furthermore, we evaluated literature in the field.
Results: An increased CPRG substrate concentration combined with a different detergent, Saponin, and an optimal wavelength recording markedly increased the sensitivity for the detection of beta-galactosidase activity (approximate to 4-fold increase). Moreover, the improved protocol resulted in increased linear time-dependent recording of enzymatic activity once cells had been lysed, and a more stable and reproducible assay to detect a ligand-stimulus with the reporter cells. The optimal time length of exposure to a stimulus was ligand-dependent.
Discussion: In conclusion, we provide an improved protocol with an optimized lysis buffer that gives up to a sixfold higher and more robust specific signal when NFAT/lacZ-based receptor-expressing reporter cells are exposed to a stimulus.

• 出版日期2018-4