Transfection of cells was done using Lipofectamine 2000 reagent (Invitrogen) per manufacturer’s instructions as explained previously [14 (link)]. Briefly, Hep3B cells were seeded and 1 day later co-transfected with 3 μg each of cmv-Gluc1-AR and cmv-JunD-Gluc2 constructs. Transfections with cmv-Gluc1-AR or cmv-JunD-Gluc2 alone were used as negative controls, and cmv-Gluc1-smad3 co-transfected with cmv-PKB-Gluc2 [19 (link)] was used as a positive control. Two to 3 hr after transfection, cells were washed and refed with DMEM without serum and either treated with 2 nmol/l R1881 (+R) or left untreated (−R). The cells were incubated after transfection at 37°C under 5% CO2 for 48 hr, then lysed using a lysis buffer provided in a Gaussia luciferase assay kit from New England Biolabs (Ipswich, MA) following the manufacturer’s protocol.
Gaussia luciferase assay kit
Manufactured by New England Biolabs
Sourced in United States
The Gaussia luciferase assay kit is a bioluminescence-based detection system designed for the quantitative analysis of Gaussia luciferase activity. Gaussia luciferase is a naturally secreted enzyme that emits light upon the addition of the substrate coelenterazine. The kit provides the necessary reagents to perform this luminescent assay.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Prism 5, by GraphPad (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Phospha light seap reporter gene assay system, by Thermo Fisher Scientific (1 mentions)
Ifn α, by Merck Group (1 mentions)
Transit lt1 transfection agent, by Mirus Bio (1 mentions)
Fluoroskan ascent fl, by Thermo Fisher Scientific (1 mentions)
Ku55933, by Selleck Chemicals (1 mentions)
0.45 μm filter, by Pall Corporation (1 mentions)
Fugene 6, by Promega (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
D luciferin, by Merck Group (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Mda mb 468, by American Type Culture Collection (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Microlumat plus, by Berthold Technologies (1 mentions)
Luciferase reporter assay system, by Promega (1 mentions)
21 protocols using gaussia luciferase assay kit
1
Transfection and Reporter Assay in Hep3B Cells
2
Firefly Luciferase Reporter Assay Protocol
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The protocol for the firefly luciferase reporter assay was described previously [18 (link)]. Briefly, 3.0 g of TCF/LEF luciferase reporter was added to culture flasks, and transfection was performed with LipofectamineTM 2000 (Invitrogen, Gaithersburg, MD, USA). Twenty-four hours after transfection, the treated cells were replanted in 24-well plates, and EGCG or DMSO was then added to the cells in each group. After 24 hours, the upper medium was collected for fluorescence activity detection using a Gaussia Luciferase Assay kit (New England Biolabs).
3
Luciferase Assay for Monitoring MondoA Activity
4
Binding Affinities of Mouse TNF and STAR2 to TNFR1/2
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To analyze the binding of wild-type mouse TNF and mouse STAR2 to mouse TNFR2, HEK293 cells were electroporated with an empty vector or a mouse TNFR2 expression plasmid. The next day, transfected cells were seeded into 24-well plates. After an additional day, the cells were incubated pairwise at 37°C for 2 h with increasing concentrations of GpL-TNC–mouse TNF and GpL-STAR2, and cell-bound luciferase activity was assayed using the Gaussia luciferase assay kit (New England Biolabs, Inc.) on a Lucy 2 luminometer (Anthos Labtec Instruments). Specific binding values were calculated for the two GpL fusion proteins by subtracting the values obtained for empty vector–transfected cells (nonspecific binding) from the corresponding values verified for the TNFR2 transfectants. To analyze the binding of wild-type mouse TNF and STAR2 to mouse TNFR1, a black high-bond 96-well ELISA plate coated with 0.5 µg/ml protein G was loaded with 1 µg/ml of an Fc fusion protein of the ectodomain of mouse TNFR1. Wells were then incubated with increasing concentrations of GpL–mouse TNF and GpL-STAR2 for 1 h at 37°C, and the well-associated luciferase activity was quantified using the Gaussia luciferase assay kit. Bmax and Kd values were obtained by fitting the binding data by nonlinear regression to a one side–specific binding plot using Prism5 (GraphPad Software).
5
Time-of-Drug-Addition Assay for HCV Infection
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The time-of-drug-addition assay was performed using an earlier described method22 (link), by adding DHMD (50 μM) to Huh-7.5 cell monolayers (1 × 104 cells/well of a 96-well plate) either 24 h before, concurrently with, or subsequent to HCVcc inoculation (multiplicity of infection [MOI] = 0.01). Wash steps were included to ensure that the drug was only present during the specific treatment period (Fig. 3a ). Following 72 h of incubation, the supernatant was collected and luciferase reporter signals reflecting viral infectivity was determined using the Gaussia luciferase assay kit (New England Biolabs; Pickering, ON, Canada) and a luminometer (Promega; Madison, WI, USA) as previously published20 (link). Data are expressed as log10 of relative light units (log10 RLU). For comparison, IFN-α (1,000 international unit [IU]/ml; Sigma) and DMSO (0.5%) served as positive and negative control treatments, respectively.
6
Gaussia Luciferase Assay in 293T Cells
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A total of 1μg DNA (200 ng per construct) was transfected into 293T cells using TransIT-LT1 transfection agent (MirusBio, Madison, WI, USA) according to the manufacturer’s recommendations. As a negative control, pPOL1-Luc plasmid alone was used to transfect into the cells. The assay was performed at 37 °C. Supernatants of transfected cells were harvested at fixed time points post-transfection and luciferase activities were measured using a Gaussia Luciferase Assay Kit (NEB), and the signals were read using a Fluoroskan Ascent FL (ThermoFisher Scientific, Waltham, MA, USA).
7
Measuring Albumin Promoter-Driven Luciferase Activity
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Prior to induction, HP14.5d cells (8×104) were seeded in 24-well culture plates at an initial confluence of 30% and transfected with a homemade plasmid containing an albumin (ALB) promoter-driven luciferase reporter gene (pSEB-ALB-Gluc), using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as the transfection reagent (15 (link)). Briefly, the ALB promoter was amplified by polymerase chain reaction and inserted into the multi-cloning site of a pBGLuc vector, as previously described (14 (link),15 (link)). The sequence of the pBGLuc plasmid sequence can be accessed at: http://www.boneandcancer.org/MOLab%20Vectors%20after%20Nov%201%202005/pBGLuc.pdf . At the indicated time points, culture medium was collected and GLuc activity was assayed using the Gaussia Luciferase Assay kit (New England Biolabs, Inc., Ipswich, MA, USA). All measurements were performed in triplicate.
8
Evaluating ATM and MRN Regulation of rAAV Expression
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To detect the effect of ATM on rAAV expression, BHK21 cells were seeded at 30% confluence in each well of a six-well plate and treated with 10 μM ATM inhibitor (KU55933; Selleck, USA) or 10 μM dimethyl sulfoxide (DMSO) on day 1, and the cells were infected with rAAV8wt-Gluc and rAAV8biΔBC-Gluc on day 2, respectively. Gluc activity was measured 48 h post-infection with the Gaussia Luciferase Assay kit (New England Biolabs) according to the manufacturer’s instructions. Briefly, 50 μL of substrate solution was added to 20 μL of culture medium and RLU were determined using a luminometer. Cells were maintained in the presence of the ATM inhibitor or DMSO until Gluc activity was assayed. To detect the effect of the MRN complex on rAAV expression, BHK21 cells were seeded and infected with rAAV as described above, but treated with 25 μM Mre11 inhibitor (Mirin, Sigma, USA) or 25 μM DMSO.
The effect of MRN degradation on AAV expression was also evaluated. HEK293 cells were seeded at 30% confluence in each well of a six-well plate, and transfected with pHelper plasmids 48 h later. The cells were infected with rAAV8wt-Gluc and rAAV8biΔBC-Gluc after counting, 12 h post-transfection. Finally, 48 h post-transfection, Gluc activity was measured as described above.
The effect of MRN degradation on AAV expression was also evaluated. HEK293 cells were seeded at 30% confluence in each well of a six-well plate, and transfected with pHelper plasmids 48 h later. The cells were infected with rAAV8wt-Gluc and rAAV8biΔBC-Gluc after counting, 12 h post-transfection. Finally, 48 h post-transfection, Gluc activity was measured as described above.
9
Generating Engineered HIV-1 Viruses
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Viruses carrying wild-type, D368R mutant HIV-1 Env were generated by transfection of HEK293 cells with full-length WT or mutant pNL4-3 or Q23_BG505 constructs, or at a 1:1 ratio of pNL4-3 ΔEnv and WT or mutant pCAAGS_JR-FL, along with 1:6 ratio of HIV-1-InGluc to viral constructs, using Fugene 6 (Promega, Madison, WI). Supernatants from 24 and 48 hr post-transfection were combined, filtered through a 0.45 μM filter (Pall Corporation) and titered on TZMbl cells. Titers were determined 48 hr post-infection either by measuring luciferase activity using the Gaussia luciferase assay kit (NEB). For testing the effects of ligands on infectivity, viruses were incubated with ligands for 30 min at room temperature prior to addition to TZMbl cells.
The V1V4-tagged BG505 was validated in form of the 100% tagged virus, not as the dye-labeled virus since the incomplete labeling efficiencies would leave enough unmodified trimers on the surface of a virus to account for nearly undiminished infectivity. While we cannot exclude additional effects of the dyes, we have not observed anisotropy for singly labeled virus, the dyes are highly hydrophilic (Zheng et al., 2014 (link)), do not associate with the viral membrane and no dye associated with viruses in the absence of labeling tags (Munro et al., 2014 (link)).
The V1V4-tagged BG505 was validated in form of the 100% tagged virus, not as the dye-labeled virus since the incomplete labeling efficiencies would leave enough unmodified trimers on the surface of a virus to account for nearly undiminished infectivity. While we cannot exclude additional effects of the dyes, we have not observed anisotropy for singly labeled virus, the dyes are highly hydrophilic (Zheng et al., 2014 (link)), do not associate with the viral membrane and no dye associated with viruses in the absence of labeling tags (Munro et al., 2014 (link)).
10
Cell-Based Luciferase Assay Protocol
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All restriction enzymes, T4 ligase and T4 polynucleotide kinase were purchased from Fermentas (Thermo Scientific). Fetal bovine serum (FBS), Opti-MEM and 0.25% trypsin-EDTA were obtained from GIBCO. Tetracycline-screened FBS, antibiotic-free high-glucose Dulbecco's modified Eagle's medium (DMEM) and 10 000 U/ml penicillin and 10 000 μg/ml streptomycin were purchased from HyClone. Lipofectamine 2000 was purchased from Invitrogen. The Gaussia luciferase assay kit was purchased from NEB. D-Luciferin was purchased from Sigma-Aldrich. Human embryonic kidney 293 cells (HEK293), human cervical carcinoma cells (HeLa) and MDA-MB-468 cells were obtained from ATCC. Four-week-old male ICR mice (≈20 g body weight) were purchased from SLRC Laboratory Animals.
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