All chemicals were purchased from Sigma unless otherwise indicated. The ISD was prepared by mixing equimolar amounts of sense and antisense DNA oligonucleotides [Sense strand 5′-TACAGATCTACTAGTGATCTATGACTGATCTGTACATGATCTACA-3′]. The oligos were heated at 95 °C (5 mins) and then cooled to RT. Poly(I:C), Poly(DA-DT) and cGAMP was purchased from Invivogen. Puromycin, blasticidin, zeocin, Quanti-Luc (InvivoGen), kanamycin (Sigma-Aldrich) and hygromycin (Roche).
Quanti luc
Manufactured by InvivoGen
Sourced in France, United States
QUANTI-Luc is a bioluminescent assay kit designed for the quantitative measurement of luciferase reporter gene expression. It provides a sensitive and reliable method for detecting and quantifying luciferase activity in cell lysates or culture supernatants.
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Lab products found in correlation
Quanti blue, by InvivoGen (16 mentions)
Zeocin, by InvivoGen (9 mentions)
Normocin, by InvivoGen (8 mentions)
Jurkat lucia nfat cd16 cells, by InvivoGen (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Quanti blue solution, by InvivoGen (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Cgamp, by InvivoGen (2 mentions)
Blasticidin, by InvivoGen (2 mentions)
Raw lucia isg mda5 ko, by InvivoGen (2 mentions)
Raw lucia isg rigi ko, by InvivoGen (2 mentions)
Raw lucia, by InvivoGen (2 mentions)
Poly 1 c, by InvivoGen (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Nsg mice, by Charles River Laboratories (2 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (2 mentions)
Hepg2 lucia ahr reporter cells, by InvivoGen (2 mentions)
84 protocols using quanti luc
1
Preparation and Characterization of ISD
2
CD16 Activation Reporter Assay
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A CD16 activation reporter assay was performed as previously described62 . Briefly, the wells of a high-binding 96 well plate were coated with 1 μg/mL recombinant gD protein in PBS and incubated overnight at 4°C. The plate was washed 3x with 1x PBS with 0.01% Tween20 and blocked at room temperature with 1x PBS with 2.5% BSA for 1 hour. Antibodies were serially diluted in growth medium and added to the washed plate with 100,000 Jurkat Lucia NFAT CD16 cells/well (Invivogen). Antibodies and cells were incubated for 24 hours at 37°C with 5% CO2. A 25 μL volume of the cell supernatant was removed and added to a new, opaque white 96 well plate. A 75 μL volume of the QuantiLuc (Invivogen) substrate was added to the supernatant and luminescence was immediately read on SpectraMax Paradigm plate reader (Molecular Devices) using a 1 second integration time. A kinetic read time of 0, 2.5 and 5 minutes was performed, and the reported values are the averages of the three reads. Buffer only wells were used as negative controls and a cell stimulation cocktail with 2 μg/mL ionomycin was used as a positive control. The assay was performed in technical replicate.
3
SEAP and Lucia Assays for cGAMP and DNA Sensing
4
NF-kB Pathway Modulation by Blackberry Anthocyanins
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The question, whether the NF-κB signaling pathway is affected by blackberry anthocyanins or the degradation product PGA was addressed by applying a NF-κB reporter gene assay. After differentiation into macrophages, NF-κB-inducible Luc reporter cells (THP1-Lucia™ NF-κB cells) were pre-incubated for 2 h with Cy3glc or PGA (0.005–10 µM) and subsequently stimulated with LPS (10 ng/mL) for 18 h, as recommended in the manufacturer’s protocol to induce luciferase gene expression by activation of NF-κB. The quantification of secreted Lucia luciferase in cell culture media is based on the catalytic oxidation of the coelenterazine-based luminescence assay reagent (QUANTI-Luc™) which leads to the emission of visible blue light (λ = 465–493 nm). For this, an aliquot of luciferase containing supernatant was transferred (10 µL) into a 96-well plate and fully automated 50 µL of QUANTI-Luc™ (Invivogen, Toulouse, France) were injected into each well with a dispenser. The resulting luminescence was measured with the Gen5 Microplate Reader (BioTek Instruments).
5
Quantifying NF-κB and IRF Induction
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THP1-Dual cells were pretreated with WE or indicated chemicals for 6 h, then stimulated with TLR2 ligand (0.1 μg/ml Pam3CSK4) or RIG-I/MDA5 ligand (0.05 μg/ml poly(I:C)/LyoVec), following the manual’s procedures. After undergoing stimulation for 24 h, NF-κB and IRF induction was quantified by measuring the levels of secreted alkaline phosphatase (SEAP) and luciferase activity in the culture supernatant using QUANTI-Blue (InvivoGen) and QUANTI-Luc (InvivoGen), respectively. Changes from baseline were calculated as -fold changes, normalized against unstimulated control cells.
6
Quantitative Lucia Luciferase Assay
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The 450 bp upstream ARE of SGK1 was cloned into the CpG-free lucia promoter vector (Invivogen, Cat# pcpgf-prom). As the normalizing control, the pSELECT-zeo-SEAP vector (Invivogen, Cat# psetz-seap) was used. 3 × 105 cells were seeded in 6-well plates to ensure cells reach ~40% confluency in 24 h. Transfection using LipoD293 reagent (SignaGen, Cat# SL100668) was used according to the manufacturer's instructions. Briefly, using a 96-well format, 0.25 μg of plasmid (both the lucia test plasmid and the SEAP control plasmid) and 0.6 μl of LipoD293 reagent were used for each transfection, and transfections were performed in biological triplicates. To detect both secreted lucia and secreted alkaline phosphatase, Quanti-luc (Invivogen, Cat# rep-qlc1) and Quanti-Blu (Invivogen, Cat# rep-qb1) were used respectively, according to manufacturer's instructions. Lucia was measured using the Lmax luminometer machine and SEAP was measured after overnight incubation at 37°C. Statistical significance was determined using the two sided Student's T-test.
7
Quantifying Luciferase Assay Protocols
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Supernatant from each sample was collected and immediately used in the luciferase assay. Luciferase presence in the supernatant was quantified by kit (QUANTI-luc, InvivoGen, catalog number rep-qlc1). Each sample was tested in triplicate in white 96-well plates with clear bottoms (LUMITRAC 200, Greiner Bio-One, Monroe, NC) with 5 times of volume to the supernatant sample. For each sample in triplicate the arithmetic average was reported. Fold induction is calculated as previously reported [6 (link)].
8
ISRE-IRF3 Promoter-Reporter Assay
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To evaluate the activity of stably expressed ISRE-IRF3 promoter-reporter, RAW Lucia and IRF3KO RAW Lucia cells were challenged with TMEV, or stimulated with 10 μg/ml poly I:C with or without 20 ng/ml IFN-γ, (Guinn and Petro, 2019 (link)) or stimulated with 20 ng/ml IL-33 for 18-24 h. Ten μl of supernatant was mixed with 50 μl of QuantiLuc (Invivogen) in a 1.5 ml microcentrifuge tube and relative luminescence was immediately determined using a Turner Biosystems Luminometer TD20/20.
9
Quantifying IRF Activation in M. tuberculosis Infection
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RAW-Lucia-ISG, RAW-Lucia-ISG-MDA5-KO, and RAW-Lucia-ISG-RIGI-KO (InvivoGen) cells were generated from the murine RAW 264.7 macrophage cell line by stable integration of a secreted Lucia luciferase reporter under the control of an ISG54 minimal promoter and 5 IFN-stimulated response elements, thus allowing IRF activation to be monitored by levels of Lucia luciferase in the cell culture supernatant. In brief, the cells were inoculated with the M. tuberculosis H37Rv strain at an MOI of 5 or 20 for 2 hours and then washed with DMEM and further cultured in complete DMEM. After 24 hours of incubation, supernatants were collected and assayed for the level of IRF induction by determination of the activity of Lucia luciferase using QUANTI-Luc (InvivoGen) according to the manufacturer’s protocol.
10
Quantifying Type I Interferon Activation
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RAW-Lucia ISG cells and THP1-Dual ISG cells were used to determine IRF pathway activation by the indicated formulations. Cells (180 μL) were inoculated in 96 well plates (100,000 cells/well) with DMEM and RPMI-1640 medium, respectively, and incubated for 24 h at 37 °C in 5% CO2. Then, cells were stimulated for 18 h at 37 °C in a 5% CO2 incubator with 20 μL of various cDNs-containing formulations. The quantity of type I interferon was indirectly quantified using QUANTI-Luc (InvivoGen), which was prepared and used according to the manufacturer's instructions.
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