Quanti luc assay

Manufactured by InvivoGen

Sourced in United States

The QUANTI-Luc assay is a luminescence-based reporter gene assay used to quantify luciferase activity. It provides a rapid and sensitive method for the detection and measurement of luciferase expression in cell-based models.

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Lab products found in correlation

Quanti blue assay, by InvivoGen (2 mentions) Opti mem, by Thermo Fisher Scientific (2 mentions) Tlrl picw, by InvivoGen (2 mentions) Lipofectamin 3000, by Thermo Fisher Scientific (1 mentions) Infinite 200 pro reader, by Tecan (1 mentions) 96 well flat bottom plate, by Corning (1 mentions) 5 ppp dsrna, by InvivoGen (1 mentions) Tl8 506, by InvivoGen (1 mentions) 2 3 cgamp, by InvivoGen (1 mentions) Synergy h1 plate reader, by Agilent Technologies (1 mentions) 3 ppp hprna, by InvivoGen (1 mentions) Envision multilabel reader, by PerkinElmer (1 mentions) Recombinant ifn γ, by BioLegend (1 mentions) Recombinant ifn β, by R&D Systems (1 mentions) Qiafilter plasmid maxi kit, by Qiagen (1 mentions) Poly 1 c, by InvivoGen (1 mentions) Tlrl 3prna, by InvivoGen (1 mentions) 5 triphosphate dsrna, by InvivoGen (1 mentions) Isotype control antibody, by BioLegend (1 mentions) Ifn γ neutralizing antibody, by BioLegend (1 mentions)

Spelling variants of this product

QUANTI-Luc (84 citations) QUANTI-Luc assay solution (4 citations) QUANTI-Luc coelenterazine-based luminescence assay detection reagent (2 citations) QUANTI-Luc luminescence assay reagent (2 citations)

10 protocols using quanti luc assay

Optimization of HACS Activity Assay in Multiple Cell Lines

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QM9, DF-1, A549, ST or LoVo cell cultures in 24-well plates were transfected at 70% confluency using Lipofectamin 3000^® (Thermo Fisher Scientific) according to the manufacturer’s instructions. At 48 h after transfection, cell-free culture supernatant was collected. Transfection efficiency of the different cell lines was controlled by fluorescence microscopy using a plasmid of comparable size and expressing EGFP.
Chemoluminescence activity in collected supernatants was determined using the QUANTI-Luc™ assay (InvivoGen, USA). In brief, 15 µL of the respective cell-free supernatant were pipetted in triplicate to which 50 µL per well of the QUANTI-Luc™ reagent were added and immediately measured in the Infinite 200 PRO reader (TECAN). Comparison between different cells, transfection efficiencies and cleavage sites were facilitated by use of a negative control (BACE-only, no HACS) for normalization. Fold-increase values were calculated on that base. The two-sample t-test was used to evaluate the average difference between each of two HACS configurations.

Parvin R., Schinkoethe J., Grund C., Ulrich R., Bönte F., Behr K.P., Voss M., Samad M.A., Hassan K.E., Luttermann C., Beer M, & Harder T. (2020). Comparison of pathogenicity of subtype H9 avian influenza wild-type viruses from a wide geographic origin expressing mono-, di-, or tri-basic hemagglutinin cleavage sites. Veterinary Research, 51, 48.

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Dual Reporter Assay for Innate Immunity

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THP1-Dual, THP1-Dual MyD88^−/−, and THP1-Dual MAVS^−/− cells were seeded in 96-well V-bottom culture plates (Corning) at 75,000 cells per well in antibiotic-free RPMI 1640 growth media with 10% HI-FBS for 3 hours before transfection. A549-Dual and A549-Dual RIG-I^−/− cells were seeded in 96-well flat-bottom plates (Corning) at 20,000 cells per well in antibiotic-free DMEM with 10% HI-FBS for 24 hours before transfection. Cells were transfected with mRNA (250 ng per well) or 3′ppp-hpRNA (2500 ng per well; InvivoGen), 5′ppp-dsRNA (2500 ng per well; InvivoGen), or 2′3′-cGAMP (2500 ng per well; InvivoGen) or treated with agonists R848 (2500 ng per well; InvivoGen) or TL8-506 (2500 ng per well; InvivoGen) for 24 hours. Luciferase in the supernatant was measured by the QUANTI-Luc assay (InvivoGen), and SEAP was measured by the QUANTI-Blue assay (InvivoGen) on a Synergy H1 plate reader (BioTek).

Nelson J., Sorensen E.W., Mintri S., Rabideau A.E., Zheng W., Besin G., Khatwani N., Su S.V., Miracco E.J., Issa W.J., Hoge S., Stanton M.G, & Joyal J.L. (2020). Impact of mRNA chemistry and manufacturing process on innate immune activation. Science Advances, 6(26), eaaz6893.

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RNA Transfection and Omics Analysis

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All RNAs and scrambled negative control dsRNA were synthesized by Integrated DNA Technologies, lnc. (IDT). The poly(I:C) was purchased from InvivoGen (Cat# tlrl-picw), which specifically confirmed the absence of contamination by bacterial lipoproteins or endotoxins. Cells were seeded into 6-well plate at 3 × 10⁵ cells/well or 96-well plate at 10⁴ cells/well and cultured for 24 h before transfection. Transfection was performed using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions with some modifications. If not indicated otherwise, 6.8 μL of 10 μM RNA stock solution and 5 μL of transfection reagent were added in 200 μL Opti-MEM (Invitrogen) to make the transfection mixture. For transfection in 6-well plate, 200 μL of the transfection mixture was added to each well; for transfection in 96-well plate, 10 μL of the transfection mixture was added to each well. At indicated times after transfection, cell samples were collected and subjected to RNA-seq (Genewiz, lnc.), TMT Mass spectrometry, qRT-PCR, western blot, or Quanti-Luc assay (InvivoGen).

Si L., Bai H., Oh C.Y., Zhang T., Hong F., Jiang A., Ye Y., Jordan T.X., Logue J., McGrath M., Belgur C., Nurani A., Cao W., Prantil-Baun R., Gygi S.P., Powers R.K., Frieman M., tenOever B.R, & Ingber D.E. (2021). Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity. bioRxiv.

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Interferon-Responsive Luciferase Reporting

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Human THP-1 or murine RAW264.7 cells containing an interferon regulatory factor (IRF)-inducible luciferase reporter were seeded at 2 × 10e4 cells per well in 96-well plates. After overnight incubation, cells were stimulated with the indicated compound for 24 h. Luciferase activity in the supernatant was measured using Quanti-Luc assay (Invivogen) and an Envision reader (PE) according to manufacturer's instructions.

Zhu Q., Hu H., Liu H., Shen H., Yan Z, & Gao L. (2020). A synthetic STING agonist inhibits the replication of human parainfluenza virus 3 and rhinovirus 16 through distinct mechanisms. Antiviral Research, 183, 104933.

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Quantifying Doxycycline-Induced CXCR4 Expression

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H1299-IL24 cells (1x10⁵) seeded in six-well tissue culture plates were transiently transfected with 2 μg of pORF9-hCXCR4 Quanti-Luc plasmid (InvivoGen, San Diego, CA, USA) encapsulated in cationic DOTAP:Cholesterol liposome [30 (link)]. After 6 h of transfection, tissue culture medium was removed and replenished with fresh medium supplemented with or without doxycycline (1 μg/ml). At 24 h after doxycycline treatment, 10 μl of culture supernatant was taken from each sample and transferred to a 96-well white (opaque) plate and 50 μl of Quanti-Luc assay (InvivoGen) reagent was added and luciferase activity was measured by Perkin Elmer EnVision Multilabel Reader (Waltham, MA, USA), according to the manufacturer’s instruction. The results from duplicate wells for each sample was calculated and represented as the average of duplicate samples. Experiments were performed independently for a minimum of three times for calculating statistical significance.

Panneerselvam J., Jin J., Shanker M., Lauderdale J., Bates J., Wang Q., Zhao Y.D., Archibald S.J., Hubin T.J, & Ramesh R. (2015). IL-24 Inhibits Lung Cancer Cell Migration and Invasion by Disrupting The SDF-1/CXCR4 Signaling Axis. PLoS ONE, 10(3), e0122439.

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Quantifying Type I Interferon Responses

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Supernatants from T cell / DC cell cultures stimulated with anti-CD3/anti-CD28 without poly I:C were diluted 1:4 in fresh media. Supernatants from T cell/DC cell cultures with anti-CD3/anti-CD28 with poly I:C were diluted 1:8. These diluted supernatants were then incubated with 10⁵ RAW-Lucia or IRF3KO-RAW-Lucia cells in culture media for 24 h. Additionally, serial dilutions of recombinant IFN-γ (Biolegend 575302) were added to 10⁵ RAW-Lucia or IRF3KO RAW-Lucia cells. Recombinant IFN-β (250 pg/ml) (R&D 124001-1) and PolyI:C (25ug/ml) were also used as positive controls for responses of RAW-Lucia and IRF3KO-RAW-Lucia cells. To assess secreted luciferase at 24 h, 10 µl of supernatants were assayed using the QUANTI-Luc assay of InVivogen.

Guinn Z., Lampe A.T., Brown D.M, & Petro T.M. (2016). Significant role for IRF3 in both T cell and APC effector functions during T cell responses. Cellular immunology, 310, 141-149.

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Quantification of IRF Pathway Activation

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THP-1-dual reporter cells were stably integrated with two inducible reporter constructs allowing the activation of the NF-kB or IRF pathways to be detected via measurement of secreted alkaline phosphatase or luciferase activity, respectively. At 24 h after poly(I:C) (10 µg/ml) stimulation, secreted luciferase in supernatant from the reporter cells was quantified by the QUANTI-Luc assay (InvivoGen) using a MicroBeta luminescence counter (PerkinElmer Wallac).

Lee S.M., Yip T.F., Yan S., Jin D.Y., Wei H.L., Guo R.T, & Peiris J.S. (2018). Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10. Frontiers in Immunology, 9, 516.

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Plasmid Construction for Secretable Luciferase Assay

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Plasmids were constructed based on the pSELECT-N-Lucia system (InvivoGen, USA) as described previously [53 (link)]: A secretable luciferase reporter (qLUC) was linked to a trans-Golgi network (TGN) anchor sequence. As a linker between these two sequences, the hemagglutinin cleavage site (HACS) region of A/chicken/Belgium/AI1940/19 [H3N1] (LATGMRNVPEKQTK*GLFGA) was introduced in frame. As a positive control, a construct expressing a polybasic HACS sequence (LATGLRNSPQRERRRKR*GLFGA) derived from the HPAIV subtype H5N1 (A/chicken/Bangladesh/AR134-c1/2016 [H5N1, clade 2.3.2.1a]) was used. Another construct lacking any HACS served as a negative control. Sequence identity was confirmed by Sanger sequencing, and plasmids were purified by the QIAfilter Plasmid Maxi kit (Qiagen) before transfection for protein expression. qLUC is only released and secreted into the culture medium if endoproteolytic processing has been accomplished. Japanese quail fibroblasts [QM9 (CCLV-RIE 466: CVCL-0I49)], and a human pneumocyte-II lineage [A549 (ATCC) CCL-185] were transfected and chemoluminescence activity in collected supernatants was determined using the QUANTI-Luc assay (InvivoGen, USA) according to the previously described protocol.

Schön J., Breithaupt A., Höper D., King J., Pohlmann A., Parvin R., Behr K.P., Schwarz B.A., Beer M., Stech J., Harder T, & Grund C. (2021). Neuraminidase-associated plasminogen recruitment enables systemic spread of natural avian Influenza viruses H3N1. PLoS Pathogens, 17(4), e1009490.

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Transfection and Downstream Analyses

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All RNAs and negative control dsRNA were synthesized by Integrated DNA Technologies (IDT). The poly(I:C) was purchased from InvivoGen (catalog no. tlrl-picw), which specifically confirmed the absence of contamination by bacterial lipoproteins or endotoxins. The 5′ triphosphate dsRNA (catalog no. tlrl-3prna) was purchased from InvivoGen. Cells were seeded into a six-well plate at 3 × 10⁵ cells/well or 96-well plate at 10⁴ cells/well and cultured for 24 h before transfection. Transfection was performed using TransIT-X2 Dynamic Delivery System (Mirus) according to the manufacturer’s instructions, with some modifications. If not indicated otherwise, 6.8 μL of 10 μM RNA stock solution and 5 μL of transfection reagent were added in 200 μL of Opti-MEM (Invitrogen) to make the transfection mixture. For transfection in the six-well plate, 200 μL of the transfection mixture was added to each well; for transfection in the 96-well plate, 10 μL of the transfection mixture was added to each well. At indicated times after transfection, cell samples were collected and subjected to RNA-seq (Genewiz), TMT mass spectrometry, qRT-PCR, western blot, Quanti-Luc assay, and Quanti-Blue assay (InvivoGen).

Si L., Bai H., Oh C.Y., Jiang A., Hong F., Zhang T., Ye Y., Jordan T.X., Logue J., McGrath M., Belgur C., Calderon K., Nurani A., Cao W., Carlson K.E., Prantil-Baun R., Gygi S.P., Yang D., Jonsson C.B., tenOever B.R., Frieman M, & Ingber D.E. (2022). Self-assembling short immunostimulatory duplex RNAs with broad-spectrum antiviral activity. Molecular Therapy. Nucleic Acids, 29, 923-940.

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Luciferase Assay for Cytokine Signaling

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Supernatants from T cell / DC cultures were diluted 1:4 in culture media and incubated with either PBS, 250 ng/ml IFN-γ neutralizing antibody (Clone: R4-6A2; Biolegend 505705), or 250 ng/ml isotype control antibody (Biolegend 400413) at 37°C for 90 min prior to incubation with 10⁵ RAW-Lucia cells for 24 hours. 10ul of supernatants were assayed for secreted luciferase using the QUANTI-Luc assay from InVivogen.

Guinn Z., Lampe A.T., Brown D.M, & Petro T.M. (2016). Significant role for IRF3 in both T cell and APC effector functions during T cell responses. Cellular immunology, 310, 141-149.

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