| Id |
Subject |
Object |
Predicate |
Lexical cue |
| T1 |
156-815 |
BACKGROUND |
denotes |
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. |
| T2 |
825-1148 |
METHODS |
denotes |
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. |
| T3 |
1158-1683 |
RESULTS |
denotes |
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. |
| T4 |
1696-1923 |
CONCLUSIONS |
denotes |
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. |
