Bright glo reporter assay system

Manufactured by Promega

The Bright-Glo Reporter Assay System is a luminescent reporter assay that measures firefly luciferase activity. It provides a sensitive and stable luminescent signal for the quantitative analysis of gene expression in cell-based assays.

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Lab products found in correlation

Mab1835, by R&D Systems (1 mentions) Pmir report vector, by Thermo Fisher Scientific (1 mentions)

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Bright-Glo (240 citations) Bright-Glo system (12 citations)

2 protocols using bright glo reporter assay system

Evaluating Milk-Derived EV TGF-β Signaling

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Murine fibroblasts (NIH-3T3; ATCC CRL-1658) cells were seeded and rested for 24h prior to transduction with adenoviral CAGA₁₂-luc construct (kindly provided by Peter ten Dijke, Dept. Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands). The CAGA₁₂-boxes in the vector are transcribed by pSmad3/4 through active TGF-β receptor signaling, resulting in luciferase activity [34 (link)]. After another 24h, cells were starved for 6h in serum-free medium. After serum starvation, cells were stimulated for at least 20h with milk-derived EVs (20–200μg/ml), or recombinant human TGF- β 1 as control. Cells were lysed and luciferase activity was measured in lysates by Bright-Glo Reporter Assay System (Promega, Leiden, The Netherlands) in a luminescence microplate reader (BMG, Isogen life science, De Meern, The Netherlands). To validate the specificity for TGF- β, cells were incubated in the presence of 2,5μg/ml mouse anti-TGF- β 1,2,3 (MAB1835, R&D Systems, Abingdon, UK). Data is represented as relative luciferase units (RLU).

Pieters B.C., Arntz O.J., Bennink M.B., Broeren M.G., van Caam A.P., Koenders M.I., van Lent P.L., van den Berg W.B., de Vries M., van der Kraan P.M, & van de Loo F.A. (2015). Commercial Cow Milk Contains Physically Stable Extracellular Vesicles Expressing Immunoregulatory TGF-β. PLoS ONE, 10(3), e0121123.

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Validating miR-219 targets in 293T cells

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miR-219 locus on chromosome 2 with their ~200 bp flanking sequences were amplified by PCR from mouse genomic DNA and inserted into pCIG vector (Zhao et al., 2010 (link)). Segments carrying putative miR-219 binding sites in 3 coding regions and 3′ UTRof Nfia, Nfib, Etv5 and Lingo1 were cloned into pMIR-REPORT vector (Ambion, AM5795) as described previously (Zhao et al., 2010 (link)). For mutagenesis of miR-219 binding sites, two predicted binding sites in pMir-reporter-Nfia UTR, pMir-reporter-Nfib CD, pMir-reporter-Etv5 CD&UTR, and two predicted binding sites in pMir-reporter-Lingo1 UTR from “ACAATC” to “CTGCAG” respectively, were created by using GenEdit Site-Directed DNA Mutagenesis Kit (FirstBiotech Cat # 201321). Luciferase reporter constructs were co-transfected with vectors expressing miR-219 into 293T cells by PolyJet. The pCMV-LacZ plasmid was included as a control for transfection efficiencies. Luciferase activity was assayed 48 hr after transfection using the bright-Glo reporter assay system (Promega, E2610) and FluoReporter lacZ/Galactosidase Quantitation Kit (Life Technologies, F-2905).

Wang H., Moyano A.L., Ma Z., Deng Y., Lin Y., Zhao C., Zhang L., Jiang M., He X., Ma Z., Lu F., Xin M., Zhou W., Yoon S.O., Bongarzone E.R, & Lu Q.R. (2017). miR-219 Cooperates with miR-338 in Myelination and Promotes Myelin Repair in the CNS. Developmental cell, 40(6), 566-582.e5.

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