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

Publikation: Bidrag til tidsskriftTidsskriftartikel

  • Charlotte Jendresen
  • Michael R Daws
  • Lars N G Nilsson

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 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.

METHODS: 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.

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 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.

DISCUSSION: 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.

OriginalsprogEngelsk
TidsskriftJournal of Pharmacological and Toxicological Methods
Vol/bind90
Sider (fra-til)67-75
Antal sider9
ISSN1056-8719
DOI
StatusUdgivet - 6 dec. 2017
Eksternt udgivetJa

ID: 194807295