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

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Standard

An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells. / Jendresen, Charlotte; Daws, Michael R; Nilsson, Lars N G.

I: Journal of Pharmacological and Toxicological Methods, Bind 90, 06.12.2017, s. 67-75.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Harvard

Jendresen, C, Daws, MR & Nilsson, LNG 2017, 'An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells', Journal of Pharmacological and Toxicological Methods, bind 90, s. 67-75. https://doi.org/10.1016/j.vascn.2017.11.004

APA

Jendresen, C., Daws, M. R., & Nilsson, L. N. G. (2017). An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells. Journal of Pharmacological and Toxicological Methods, 90, 67-75. https://doi.org/10.1016/j.vascn.2017.11.004

Vancouver

Jendresen C, Daws MR, Nilsson LNG. An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells. Journal of Pharmacological and Toxicological Methods. 2017 dec 6;90:67-75. https://doi.org/10.1016/j.vascn.2017.11.004

Author

Jendresen, Charlotte ; Daws, Michael R ; Nilsson, Lars N G. / An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells. I: Journal of Pharmacological and Toxicological Methods. 2017 ; Bind 90. s. 67-75.

Bibtex

@article{bbacdc8ec9094ee6a57295f64b1f712f,
title = "An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells",
abstract = "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.",
author = "Charlotte Jendresen and Daws, {Michael R} and Nilsson, {Lars N G}",
note = "Copyright {\circledC} 2017 Elsevier Inc. All rights reserved.",
year = "2017",
month = "12",
day = "6",
doi = "10.1016/j.vascn.2017.11.004",
language = "English",
volume = "90",
pages = "67--75",
journal = "Journal of Pharmacological and Toxicological Methods",
issn = "1056-8719",
publisher = "Elsevier",

}

RIS

TY - JOUR

T1 - An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells

AU - Jendresen, Charlotte

AU - Daws, Michael R

AU - Nilsson, Lars N G

N1 - Copyright © 2017 Elsevier Inc. All rights reserved.

PY - 2017/12/6

Y1 - 2017/12/6

N2 - 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.

AB - 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.

U2 - 10.1016/j.vascn.2017.11.004

DO - 10.1016/j.vascn.2017.11.004

M3 - Journal article

C2 - 29203451

VL - 90

SP - 67

EP - 75

JO - Journal of Pharmacological and Toxicological Methods

JF - Journal of Pharmacological and Toxicological Methods

SN - 1056-8719

ER -

ID: 194807295