A modification of a previously described luciferase assay was used [15 (link)]. In brief, interleukin (IL)-6-stimulated and -unstimulated MF fibrocytes were transfected with luciferase reporter constructs that contained GLI1 gene promoter fragments. Reporter constructs were generated using pGL4.17[luc2/Neo] vector (Promega, Madison, WI, USA) and primers listed in Supplementary Table S8 . The pRL-SV40 vector producing Renilla luciferase was used as an internal control. The constructs were transfected into fibrocytes using a lipofectamine transfection medium and incubated for 24 h at 37 °C. Following transfection, the fibrocytes were incubated in 2% FBS/RPMI-1640 starvation medium (Gibco) for 1 h at 37 °C and IL-6 (20 ng/mL) was added. Detection of luciferase activity was performed using the Dual-Glo system (Promega, Madison, WI, USA) and luminescence levels were measured using Monolight 3010 luminometer (BD Biosciences). The relative luciferase activity was calculated by normalizing the firefly luminescence to the Renilla luminescence.
Dual glo system
Manufactured by Promega
Sourced in United States
The Dual-Glo system is a laboratory equipment designed for simultaneous detection and measurement of firefly and Renilla luciferase activities in cell-based assays. The system provides a simple, sensitive, and reproducible method for quantifying gene expression and monitoring cell viability or cytotoxicity in a single sample.
Automatically generated - may contain errors
Lab products found in correlation
Pmirglo vector, by Promega (3 mentions)
Fugene 6, by Promega (3 mentions)
Pgl3 vector, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Monolight 3010 luminometer, by BD (1 mentions)
Pgl4.17 luc2 neo vector, by Promega (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
20 hydroxyecdysone, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Promega (1 mentions)
Renilla luciferase assay system, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Prl tk, by Promega (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase, by Promega (1 mentions)
Pgl3 basic, by Promega (1 mentions)
96 well culture plate, by PerkinElmer (1 mentions)
Phenol red free mem, by Thermo Fisher Scientific (1 mentions)
Envision microplate reader, by PerkinElmer (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
34 protocols using dual glo system
1
Luciferase Assay for GLI1 Promoter Activity
2
Evaluating miR-1278 Binding in SOCS6 and Circ_0015278
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Potential wildtype (WT) or mutant (MUT) miR-1278 binding sites in circ_0015278 and the 3' untranslated region (3' UTR) of SOCS6 were constructed into the pmirGLO vector from Promega (Madison, WI, USA). A549 and H1229 cells were co-transfected with miR-1278 mimics or miR-NC and circ_0015278 or SOCS6 luciferase reporter. After 48–72 hours, cells were harvested and used for examining the luciferase activity with the Dual-Glo system (Promega, Madison, WI, USA).
3
NFAT-Luciferase Reporter Assay for CAR T-cell Activation
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Example 2
To identify an NFAT responsive element, a cell line stably expressing an NFAT-based Luciferase expression cassette (NR2.2) was created by electroporating a linearized construct into NK-92® cells which contains a stop region, followed by 3 NFAT response elements (SEQ ID NO:4), and a minimal promoter (SEQ ID NO:5), which in the presence of activated NFAT will thus drive the production of Firefly Luciferase. A subset of these cells were then also electroporated with mRNA containing an anti-CD19 CAR (an antigen present on Sup-B15 cells, otherwise resistant to killing by NK-92® cells). These cells are represented in the left graph as ENR2.2. The cells were then plated in triplicate in the absence or presence of target cells, and incubated for periods ranging from 2.5 hours to 24 hours. At the end of the incubation period, the Step 1 reagent from a Promega DualGlo system was added to the wells to activate luciferase (by providing its substrate, luciferin). The result was then read on a SpectraMax i3x plate reader, and presented as an average with standard deviation as calculated in Microsoft Excel. The results are shown in
4
HBV Inhibition by IFI27 in HepG2 Cells
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HepG2 cells plated in a 24-well plate were transfected with HBV reporter plasmids (200 ng) along with an indicated amount of pCAGGS-HA-IFI27 plasmid (250 ng) or pCAGGS (control vector), and the pRL-TK plasmid (50 ng) was used for the control of transfection efficiency. At 48 h post-transfection, the cells were lysed and subjected to luciferase activity assay using the Dual-Glo system (Promega, Madison, WI, United States).
5
HOTAIRM1 Luciferase Reporter Assay
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Luciferase reporter assay was performed with pGL3 vectors of Promega (Madison, Wisconsin, USA), and luminescence was detected with Dual-Glo system from Promega. HOTAIRM1 binding DNA sequences were amplified by PCR, and dsDNAs were inserted into pGL3-promoter vector between SmaI and XhoI cleavage sites, which is 1 kb downstream of 3’ end of the firefly luciferase gene. The cloned vector was co-transfected with pRL-TK renilla luciferase control reporter vectors into Kasumi-1 cells. The firefly and renilla luminescence were detected in the transfected cells after 48 h using Dual-Glo®luciferase Assay System (Promega, Madison, Wisconsin, USA). The firefly luminescence was normalized to renilla luminescence under various conditions. Folding inductions were then calculated by normalizing to the pGL3-promoter control.
6
Luciferase Assay for miRNA Activity
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A recombined pMIR‐Report luciferase vector, miR‐192‐3p or miR‐NC, and pMIR‐Report control vector were co‐transfected into cells for 36 hours. The cells were lysed and subjected to luciferase activity assays by using the Dual‐Glo system (Promega, WI).
7
Copper-Induced Attacin Expression Regulation
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Briefly, ~5×105 of afore-mentioned S2 stable cells were seeded in 24-well plates the day before transfection. Subsequently, 500 ng att-luc reporter construct (firefly luciferase reporter gene driven by the promoter of the antimicrobial peptide gene Attacin) and 20 ng of actin-Renilla luciferase were transfected into these cells. Two days post transfection, cells were treated with 250 μM copper for ~3 and 5 days, respectively, to achieve Edis overexpression and knockdown. Next, cells were treated with 1 μM 20-hydroxyecdysone (Sigma) for 24 hours and subsequently left untreated or treated with PGN for 6 hours. Cell suspensions were arrayed in 384-well plates and reporter activity was measured using the Dual-Glo system (Promega). For data processing, firefly/Renilla ratio was calculated and normalized against the control sample.
8
Phosphorylation of p38 MAPK in ASE cells
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ASE cells were maintained as previously described [35 ]. For AsP38 MAPK phosphorylation studies, ASE cells were either treated with 1 μg/ml LPS alone or pretreated with 10 μM BIRB796, 10 μM SB203580, or an equivalent volume of DMSO as a control for 2 h. Luciferase reporter assays were performed using the Defensin1, Cecropin1, and Gambicin promoter-reporter plasmids as previously described [35 ]. At 24 h post-transfection, cells were treated with 100 μg/ml LPS (Sigma-Aldrich) or 2 μg/ml peptidoglycan (PGN, Escherichia coli K12) with 0.1-10 μM BIRB796 or an equivalent volume of DMSO as a control. Luciferase activity was measured 24 h post-LPS or PGN treatment with the Dual-Glo system (Promega).
9
Transfection and Luciferase Assay in HEK293 and SW480 Cells
10
RIG-I Activation and Protein Interaction Assays
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Cells were seeded into 96-well plates and, 18 h later, transfected with 50 ng DNA of IFN-β luciferase, 17 ng DNA of renilla luciferase and 33 ng DNA of RIG-I plasmid. One day after DNA transfection, cells were transfected with Poly(I:C), synthetic RNA or infected with Moraten-gfp virus at MOI 1. The following day, the luciferase assay was performed using the Dual-Glo system from Promega. Firefly luciferase values were normalized to renilla luciferase to measure transfection efficiency.
For Gaussia Luciferase-Based Complementation Assay (PCA) [33] (link), cells were seeded into 96-well plates and, 8 h later, transfected with 100 ng RIG-I-glu1 construct and 100 ng RIG-I-glu2 construct. Twenty four hours after DNA transfection, cells were transfected or not with Poly(I:C). Eighteen hours later, the luciferase assay was performed using the Renilla Luciferase Assay System (Promega). Protein-protein interaction levels were expressed in normalized luminescence ratio (NLR) according to the following formula: where glu1-A and glu2-B are the chimeric proteins, and glu1 and glu2 the empty vector coding only for the glu fragment.
For Gaussia Luciferase-Based Complementation Assay (PCA) [33] (link), cells were seeded into 96-well plates and, 8 h later, transfected with 100 ng RIG-I-glu1 construct and 100 ng RIG-I-glu2 construct. Twenty four hours after DNA transfection, cells were transfected or not with Poly(I:C). Eighteen hours later, the luciferase assay was performed using the Renilla Luciferase Assay System (Promega). Protein-protein interaction levels were expressed in normalized luminescence ratio (NLR) according to the following formula: where glu1-A and glu2-B are the chimeric proteins, and glu1 and glu2 the empty vector coding only for the glu fragment.
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