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Luciferase activity

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Luciferase activity is a measurement of the bioluminescent reaction catalyzed by the luciferase enzyme. Luciferase catalyzes the oxidation of its substrate, luciferin, in the presence of ATP, Mg2+, and oxygen, resulting in the emission of light. This light output can be quantified using a luminometer to assess luciferase activity.

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Lab products found in correlation

β galactosidase, by Roche (2 mentions) Fluostar optima luminometer, by Promega (2 mentions) Lipofectamine, by Thermo Fisher Scientific (2 mentions) 96 well plate, by Corning (2 mentions) Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions) Passive lysis buffer (plb), by Promega (2 mentions) Penicillin, by Thermo Fisher Scientific (2 mentions) Streptomycin, by Thermo Fisher Scientific (2 mentions) Pmirglo vector, by Promega (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions) Pgl3 control plasmid, by Promega (1 mentions) Lipofectamine 2000 kit, by Thermo Fisher Scientific (1 mentions) Recombinant human tgf β1, by R&D Systems (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Translt1 reagent, by Mirus Bio (1 mentions) Jetprime reagent, by Polyplus Transfection (1 mentions) Pgl3 vector, by Promega (1 mentions) Liposome 2000 reagent, by Thermo Fisher Scientific (1 mentions) Il 33, by Promega (1 mentions)

77 protocols using luciferase activity

1

Luciferase Assay for NFκB and Hex1 Activity

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Jurkat cells were transfected with the pLTR-luc and the pLTR-NFκBmut-luc luciferase reporter episomal vectors using JetPEI TM (POLYplus) method according to the manufacturer’s protocol. Twenty-four hours post-transfection, cells were mock-treated or treated with the different compounds as indicated. At 24 hours post-treatment, cells were lysed and assayed for luciferase activity (Promega) as previously described [66 (link)]. Luciferase activities were normalized with respect to protein concentrations.
HeLa cells were transiently transfected with the Hex1(-104)Luc reporter plasmid [48 (link)] by X-tremeGENE HP Transfection Reagents (Roche). At 24 hours post-transfection, cells were mock-treated or treated with the different compounds as indicated. At 24 hours post-treatment, cells were lysed and assayed for luciferase activity (Promega). Luciferase activities were normalized with respect to protein concentrations.
Darcis G., Kula A., Bouchat S., Fujinaga K., Corazza F., Ait-Ammar A., Delacourt N., Melard A., Kabeya K., Vanhulle C., Van Driessche B., Gatot J.S., Cherrier T., Pianowski L.F., Gama L., Schwartz C., Vila J., Burny A., Clumeck N., Moutschen M., De Wit S., Peterlin B.M., Rouzioux C., Rohr O, & Van Lint C. (2015). An In-Depth Comparison of Latency-Reversing Agent Combinations in Various In Vitro and Ex Vivo HIV-1 Latency Models Identified Bryostatin-1+JQ1 and Ingenol-B+JQ1 to Potently Reactivate Viral Gene Expression. PLoS Pathogens, 11(7), e1005063.
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2

IFN and NF-κB Reporters in Immune Cells

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IFN activity in supernatants from infected or stimulated cells were assayed with HL116 cells that contain a luciferase reporter under control of the IFN-inducible 6–16 promoter. Supernatants were exposed to 20,000 HL116 cells in 96 well plates for 7 h, lysed in passive lysis buffer, and scored for luciferase activity (Promega). A standard curve for IFN activity was generated from serial dilution of recombinant human IFNa2a, with HL116 cells responding in a linear range between 2 and 200 U/ml of IFN. IFN-beta in DC supernatants was also detected by ELISA (Invitrogen) according to the manufacturers instructions.
An NF-κB reporter cell line was generated by transducing HeLa cells with a lentivirus that carries firefly luciferase driven by NF-κB response element, pGreenFire1-NF-κB (System Biosciences) and selecting stable cell populations with puromycin (1 μg/ml) (Invivogen). Supernatants from infected DCs were exposed to 20,000 NF-κB reporter cells in 96 well plates for 7 h, lysed in passive lysis buffer, and scored for luciferase activity (Promega).
Johnson J.S., Lucas S.Y., Amon L.M., Skelton S., Nazitto R., Carbonetti S., Sather D.N., Littman D.R, & Aderem A. (2018). Reshaping of the Dendritic Cell Chromatin Landscape and Interferon Pathways During HIV Infection. Cell host & microbe, 23(3), 366-381.e9.
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3

miR-139-5p Binding Site Validation

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PTEN fragment containing the putative miR‐139‐5p binding site was subcloned into the pmirGLO vector (Promega) and named wild type (WT). Then, the predicted miR‐139‐5p binding site was mutated to make a mutant (MT). Two reported plasmids were co‐transfected with miR‐139‐5p mimic into HTR‐8/Svneo cells, respectively. Forty‐eight h after transfection, luciferase activity was measured (Promega).
Liu H., Wang F., Zhang Y., Xing Y, & Wang Q. (2020). Exosomal microRNA‐139‐5p from mesenchymal stem cells accelerates trophoblast cell invasion and migration by motivation of the ERK/MMP‐2 pathway via downregulation of protein tyrosine phosphatase. The Journal of Obstetrics and Gynaecology Research, 46(12), 2561-2572.
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4

Virus Capture Assay for Antibody Neutralization

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Virus capture assays were performed as previously described.31 (link) Pseudovirions expressing the JR-FL Env and vesicular stomatitis virus (VSV) G protein were produced with the pSG3ΔENV backbone in 293T cells. Plates were coated with 50 μL of mAb at 5 μg/mL for 1 h at room temperature and then blocked in PBS with 3% BSA overnight at 4 °C. Hybridoma supernatants of mAb cell cultures were incubated with pseudoviruses for 1 h before the mixtures were added to the ELISA wells, and incubated for 3 h at room temperature. Plates were washed five times with sterile PBS, overlaid with 10 000 TZM-bl cells per well and incubated for 48 h at 37 °C. Luciferase activity was determined according to the manufacturer's instructions (Promega, Madison, WI, USA). The competition percentage was reported as the reduction of the luciferase signal compared to a serum negative control.
Pan R., Chen Y., Vaine M., Hu G., Wang S., Lu S, & Kong X.P. (2015). Structural analysis of a novel rabbit monoclonal antibody R53 targeting an epitope in HIV-1 gp120 C4 region critical for receptor and co-receptor binding. Emerging Microbes & Infections, 4(7), e44-.
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5

Regulation of HMGA1 by miR-125b

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HEK293T cells were seeded in 96-well plates for the promoter luciferase reporter assay. When the cell fusion reached 60%, 100 ng of miR-125b expression vectors or NC was transfected into the cells. A mixture containing 30 ng of 3′-UTR of wild type (WT) or mutant (MT) HMGA1 mRNA was transfected into the cells. Cells were collected 48 hours after the process of transfection. After 48 hours, luciferase activity (Promega, Madison, WI, USA) was determined via a dual luciferase reporter assay system in accordance with the manufacturer’s instructions.
Sun B., Zhang Y., Zhou L., Yin L., Li F., Li C, & Xia J. (2019). The proliferation of cervical cancer is promoted by miRNA-125b through the regulation of the HMGA1. OncoTargets and therapy, 12, 2767-2776.
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6

Luciferase Assay for miR-34c-5p Binding to AREG 3'UTR

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The wild-type AREG-3′UTR was cloned into the pGL3-control plasmid (Promega). The mutant AREG-3′UTR was generated by site directed mutagenesis. The Luc-3′UTR-wt or Luc-3′UTR-mt reporter plasmids were prepared by inserting the AREG-3′UTR-wt carrying a putative miR-34c-5p binding site or its mutant sequence, respectively, into the pGL3-control plasmid. Primer sequences were described in Supplementary Table 3. Luc-AREG-3′UTR-wt or Luc-AREG-3′UTR-mt was cotransfected with the miR-34c-5p plasmid into parental SKOV-I6 and OVS1 cells. The cells were harvested 48 h after transfection. Luciferase activity was measured according to the manufacture’s protocol (Promega). Renilla luciferase was cotransfected as a control for normalization.
Tung S.L., Huang W.C., Hsu F.C., Yang Z.P., Jang T.H., Chang J.W., Chuang C.M., Lai C.R, & Wang L.H. (2017). miRNA-34c-5p inhibits amphiregulin-induced ovarian cancer stemness and drug resistance via downregulation of the AREG-EGFR-ERK pathway. Oncogenesis, 6(5), e326-.
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7

Investigating lncRNA TINCR-miR-375 Interaction

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The lncRNA TINCR was predicted to have a continuous binding site with the miR-375 nucleotide sequence using LncBase v.2. A continuous binding site with miR-375 was predicted to exist in the 3′ untranslated region (3′ UTR) of ATG7 using the bioinformatics tool TarBase v.8. Cotransfection of HepG2 cells with lncRNA TINCR-WT, lncRNA TINCR-Mut, ATG7-WT, ATG-MUT, and miR-375 mimic was performed using the Lipofectamine™ 2000 kit as per the manufacturer's instructions (Invitrogen, USA). The transfection medium was changed to fresh media 12 hours later. Cells were extracted after 48 hours. The luciferase activity was detected following the manufacturer's instructions (Promega, USA).
Tang C., Yu H., Zheng Y., Feng W, & Shen J. (2022). lncRNA TINCR Regulates Proliferation and Invasion of Hepatocellular Carcinoma Cells by Regulating the miR-375/ATG7 Axis. Journal of Oncology, 2022, 8132403.
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8

Measuring Cell-Intrinsic TGF-β Activation

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The αvβ8 ectodomain was coated along with the controls αvβ3 (R&D Systems), BSA (Sigma-Aldrich) or anti-LAP (R&D AF426, 1 μg/ml) onto ELISA plates in PBS (1mM Ca2+ and 1mM Mg2+) 1 hour at RT. Wells were subsequently washed in PBS and blocked in PBS with 1% BSA WT L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP expressing TMLC cells were plated at a density of 1×105 cells/ml in basal media. After 16 hrs, media was removed and applied to wells containing TMLC reporter cells to measure diffusible mature TGF-β, which were incubated overnight prior to lysis and determination of luciferase activity (Promega) as reported (Mu, et al, 2002 (link)). To measure cell-intrinsic TGF-β1 activation, the attached L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP or parental TMLC cells were lysed and assayed for luciferase activity. To facilitate comparison between different TMLC lines expressing WT L-TGF-β1, L-TGF-β1 (R249A), WT L-TGF-β/GARP, L-TGF-β (R249A)/GARP (or parental TMLC cells) normalized luciferase activity was expressed as activated TGF-β in pg/ml. Normalization was performed by interpolating luciferase activity against standard curves generated using each TMLC line with varying doses of recombinant human TGF-β1 (R&D Systems).
Campbell M.G., Cormier A., Ito S., Seed R.I., Bondesson A.J., Lou J., Marks J.D., Baron J.L., Cheng Y, & Nishimura S.L. (2020). Cryo-EM reveals integrin-mediated TGF-β activation without release from latent TGF-β. Cell, 180(3), 490-501.e16.
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9

Regulation of PRLR Promoter by PRL

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MCF-7A2 (MCF-7) (ER+ and HER2+) (gift from E. Berleth, C. Roswell Park Cancer Institute, New York, NY) and T47D (ER+ and HER2+) breast cancer cells obtained from ATCC were maintained in RPMI 1640 medium supplemented with 10% charcoal-treated fetal bovine serum. Cells were cultured in six-well plates under steroid-free conditions in PRPMI phenol red-free media without supplemental growth factors with 5% and 1% charcoal-treated fetal bovine serum for 2 days at each serum concentration. The PRLR hPIII promoter/non-coding exon 1 (hE13) reporter pGL2 gene construct (bp −480/−112) containing C/EBPβ, Sp1 sites in hPIII and putative STAT5 binding site in hE13, and other constructs with the sites mutated, and pGL2 empty vector were used for transient transfection in cells using Lipofectamine 2000 reagent (Invitrogen) as described previously [11 (link)]. After 18 h, cells were treated with PRL (100 ng/ml) for 6 h and harvested for the determination of luciferase activity (Promega).
Kavarthapu R., Morris C.H, & Dufau M.L. (2014). Prolactin induces up-regulation of its cognate receptor in breast cancer cells via transcriptional activation of its generic promoter by cross-talk between ERα and STAT5. Oncotarget, 5(19), 9079-9091.
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10

SARS-CoV-2 Pseudotyped Virus Neutralization Assay

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SARS-CoV and SARS-CoV-2 pseudo-typed viruses were produced as previously described.14 (link) Briefly, plasmids coding full-length S protein and pNL4-3.luc.RE were cotransfected into 293T cells in 10 cm dishes. The supernatants were harvested 48 h after transfection and diluted in complete DMEM mixed with or without an equal volume (50 μl) of diluted serum or antibody and then incubated at 37 °C for 1 h. The mixtures were transferred to HEK 293 T cells stably expressing human ACE2. The cells were incubated at 37 °C for 48 h, lysed with passive lysis buffer and tested for luciferase activity (Promega USA). The percent neutralization was calculated by comparing the luciferase value of the antibody or serum group to that of the virus-only control.
Yi C., Sun X., Ye J., Ding L., Liu M., Yang Z., Lu X., Zhang Y., Ma L., Gu W., Qu A., Xu J., Shi Z., Ling Z, & Sun B. (2020). Key residues of the receptor binding motif in the spike protein of SARS-CoV-2 that interact with ACE2 and neutralizing antibodies. Cellular and Molecular Immunology, 17(6), 621-630.
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