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

• Published in: Journal of pharmacological and toxicological methods Vol. 90; pp. 67 - 75
• Main Authors: Jendresen, Charlotte, Daws, Michael R., Nilsson, Lars N.G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2018
• Subjects: Animals; beta-Galactosidase - metabolism; Biological Assay - methods; BWZ.36; Cell Line; Chlorophenol-red-β-d-galactopyranoside; Chlorophenols - metabolism; Colorimetry - methods; CPRG; Galactosides - metabolism; Ligands; Methods; Mice; Receptor stimulation; Reporter cells; Saponins - metabolism; Sensitivity and Specificity; Signal Transduction - physiology; β-galactosidase; β-galactosidase; Chlorophenol-red-β-d-galactopyranoside; Reporter cells; CPRG; Methods; Receptor stimulation; BWZ.36
• ISSN: 1056-8719; 1873-488X
• DOI: 10.1016/j.vascn.2017.11.004

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 β-galactosidase when the chimeric receptor is stimulated. A colorimetric β-galactosidase substrate, chlorophenol-red β-d-galactopyranoside (CPRG), was used to detect enzymatic activity. Sub-optimal conditions have unfortunately extensively been reported with such reporter-based β-galactosidase assays. Here, we aimed to improve the CPRG-based colorimetric assay such that receptor ligands could be effectively screened with reporter cells. After stimulation of reporter cells, we determined β-galactosidase activity by absorbance measurement of β-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. An increased CPRG substrate concentration combined with a different detergent, Saponin, and an optimal wavelength recording markedly increased the sensitivity for the detection of β-galactosidase activity (≈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. In conclusion, we provide an improved protocol with an optimized lysis buffer that gives up to a six-fold higher and more robust specific signal when NFAT/lacZ-based receptor-expressing reporter cells are exposed to a stimulus.