Dual light assay system

Manufactured by Thermo Fisher Scientific

The Dual Light Assay system is a laboratory equipment designed to measure and analyze luminescent signals in biological samples. It is capable of detecting both bioluminescent and chemiluminescent signals, allowing researchers to perform a variety of assays that rely on these methods of detection.

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Lab products found in correlation

Nucleofector solution 5, by Lonza (2 mentions) Centro lb 960 luminometer, by Berthold Technologies (2 mentions) Centro lb 960, by Berthold Technologies (2 mentions) Luminometer centro lb960, by Berthold Technologies (1 mentions) Amaxa nucleofector kit 5, by Lonza (1 mentions) Pgl3 enhancer, by Promega (1 mentions) Dual luciferase reporter assay, by Promega (1 mentions) Passive lysis buffer (plb), by Promega (1 mentions) Nucleofector system, by Lonza (1 mentions)

6 protocols using dual light assay system

Transfection of Ode Macrophages

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Ode macrophages were transfected by electroporation using the Amaxa Nucleofector, kit V and program T -017 (Amaxa Lonza protocol). 5 × 10⁵ cells/ml were washed once with PBS at room temperature, and then cells were suspended in 100 μl of Nucleofector solution V (Amaxa). Cells were co-transfected with 2 μg of luciferase reporter plasmid (3X-TRE) and/or reporter plasmid (β-galactosidase: β-gal). After transfection, cells were suspended in fresh complete medium and incubated at 37 °C, 5% CO₂ for 24 h. Measurements of luciferase and β-galactosidase activities were performed using the Dual Light Assay system (Life Technologies) and luminometer Centro LB 960 (Berthold) according to the manufacturer’s instructions.

Haidar M., Whitworth J., Noé G., Liu W.Q., Vidal M, & Langsley G. (2015). TGF-β2 induces Grb2 to recruit PI3-K to TGF-RII that activates JNK/AP-1-signaling and augments invasiveness of Theileria-transformed macrophages. Scientific Reports, 5, 15688.

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Dual Luciferase Assay in Transfected Cells

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After transfection, the cells were suspended in fresh complete medium, incubated at 37°C and 5% CO₂ for 24 h, and lysed after 48 h. According to the manufacturer’s instructions, the luciferase and β-galactosidase activities were measured using the dual light assay system (Life Technologies) and Centro LB 960 luminometer (Berthold).

Haidar M., Tajeri S., Momeux L., Mourier T., Ben-Rached F., Mfarrej S., Rchiad Z., Pain A, & Langsley G. (2023). miR-34c-3p Regulates Protein Kinase A Activity Independent of cAMP by Dicing prkar2b Transcripts in Theileria annulata-Infected Leukocytes. mSphere, 8(2), e00526-22.

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Regulation of FOS gene by miR-196b

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miRWalk2.0 was used to identify candidate genes regulated by miR-196b and to localize putative binding sites within the 3′UTR of the FOS gene (21 (link)). PITA, RNA22, and RNAhybrid 2.2 algorithms were used to localize putative binding sites for miR-196b within the FOS promoter and 5′UTR (−1500bp to translation start site +250bp) (22 (link)–24 (link)). The FOS promoter regions and the 3′UTR of the FOS gene were amplified by PCR and inserted into the pGL3-enhancer (Promega, Madison, WI) vector or pMIR-REPORT firefly Luciferase vector system (Life Technologies), respectively. The Luciferase vectors were co-transfected with either the pMIR-REPORT β-galactosidase reporter control vector or pRL Renilla (Promega) for normalizing transfection efficiency and miR-196b mimic or control mimic (Life Technologies) using Lipofectamine 3000. Luciferase and β-galactosidase activities are determined with the Dual-Light assay system (Life Technologies) and Luciferase and Renilla activities were determined by Dual-Luciferase reporter assay (Promega). The 3xAP-1pGL3 (3xAP-1 in pGL3-basic) was a gift from Alexander Dent (Addgene plasmid # 40342) (25 (link)). The AP-1 reporter plasmid was co-transfected with pRL Renilla for normalization. Reporter assays were performed in three independent experiments.

Tellez C.S., Juri D.E., Do K., Picchi M.A., Wang T., Liu G., Spira A, & Belinsky S.A. (2016). MiR-196b is epigenetically silenced during the pre-malignant stage of lung carcinogenesis. Cancer research, 76(16), 4741-4751.

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Transfection of Ode cells via Nucleofection

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Ode cells were transfected by electroporation using the “Amaxa Nucleofector, kit V and program T-017 (Amaxa Lonza protocol). 5 × 10⁵ cells/ml were washed once with PBS at RT, then cells were suspended in 100 μl of Nucleofector solution V (Amaxa). Cells were co-transfected with 2 μg of luciferase reporter plasmid (6X-CRE), or reporter plasmid (β-galactosidase: β-gal). After transfection, cells were suspended in fresh complete medium and incubated at 37 °C, 5% CO₂ for 24 h.
The measurements of luciferase and β-galactosidase activities were performed using the Dual Light Assay system (life technologies) and luminometer Centro LB 960 (Berthold) according to the manufacturer's instructions.

Haidar M., de Laté P.L., Kennedy E.J, & Langsley G. (2017). Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria-transformed leukocytes. Bioorganic & medicinal chemistry, 26(6), 1127-1134.

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Wnt/β-Catenin Transcriptional Activation Assay

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β-Catenin–mediated transcription activation was determined using mouse L cells stably expressing SuperTOPFlash (firefly luciferase reporter driven by 7× TCF/LEF β-catenin binding sites) and β-galactosidase for normalization. Cells were seeded at 5 × 10⁴ cells per well in 96-well culture plates, incubated overnight with Wnt3a or CHIR99021 at specified concentrations, then lysed in passive lysis buffer (Promega, Madison, WI) for reporter assay. For experiments with MDC, L cells were incubated with MDC with Wnt3a or CHIR99021 for 6 h at specified concentrations. Relative luciferase activity units were measured and normalized against β-galactosidase activity using the Dual-Light assay system (Thermo Fisher) on an LB 960 Centro luminometer (Berthold Technologies, Oak Ridge, TN). All assays were performed in triplicate.

Rim E.Y., Kinney L.K, & Nusse R. (2020). β-catenin-mediated Wnt signal transduction proceeds through an endocytosis-independent mechanism. Molecular Biology of the Cell, 31(13), 1425-1436.

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Transfection Protocol via Nucleofector

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Cells were transfected by electroporation using the Nucleofector system (Lonza, Basel, Switzerland). 2.5x10⁵ cells were suspended in 100μl of nucleofector solution mix with 2μg of plasmids and subjected to electroporation using the cell line—specific programme: T-O17. After transfection, cells were suspended in fresh complete medium and incubated at 37°C, 5% CO2 for 24 h and RNA extracted after 48 h post transfections. Measurements of luciferase and β-galactosidase activities were performed using the Dual Light Assay system (Thermo Fisher scientific) and luminometer Centro LB 960 (Berthold) according to the manufacturer’s instructions.

Haidar M., Rchiad Z., Ansari H.R., Ben-Rached F., Tajeri S., Latre De Late P., Langsley G, & Pain A. (2018). miR-126-5p by direct targeting of JNK-interacting protein-2 (JIP-2) plays a key role in Theileria-infected macrophage virulence. PLoS Pathogens, 14(3), e1006942.

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