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Multiplexing fluorogenic esterase-based viability assay with luciferase assays.

ABSTRACT: Luciferase-based reporter assays are one of the most common cell-based screening formats for drug discovery, and simultaneous evaluation of the cytotoxic effect of test compounds is of great value in reducing false-positives. Here we share a multiplex assay protocol that allows sequential measurement of cell viability (cell number) and luciferase activity of the same sample in a multi-well-plate format. The viability assay employs a fluorogenic esterase substrate, CytoRed. •This protocol allows sequential measurement of endogenous esterase activity (as a surrogate for cell number) and then luciferase activity in a single sample.•The protocol eliminates the need for parallel viability assay or protein assay using separate aliquots of the lysate.•This protocol is especially useful for assays with cells stably expressing a luciferase construct, for which co-transfection of another reporter gene is not a viable option.

SUBMITTER: Ohgane K

PROVIDER: S-EPMC6812399 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Multiplexing fluorogenic esterase-based viability assay with luciferase assays.

Ohgane Kenji K Yoshioka Hiromasa H Hashimoto Yuichi Y

MethodsX 20190912

Luciferase-based reporter assays are one of the most common cell-based screening formats for drug discovery, and simultaneous evaluation of the cytotoxic effect of test compounds is of great value in reducing false-positives. Here we share a multiplex assay protocol that allows sequential measurement of cell viability (cell number) and luciferase activity of the same sample in a multi-well-plate format. The viability assay employs a fluorogenic esterase substrate, CytoRed. •This protocol allows ...[more]

PMID: 31667098

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