Vero cells (ATCC) were transduced with a lentivirus expressing NanoLuc® luciferase (Vero-NLucP cells) as described previously9 . Vero-NLucP cells were plated (5000 cells/well) in a white clear-bottom 96-well plate and treated with toxin the following day. Following overnight incubation (20 h), luminescence signal was developed using the NanoGlo® Luciferase Assay kit (Promega) and read with a Spectramax M5e plate reader (Molecular Devices). Relative luminescence units (RLU) were normalized relative to untreated cells and data was analyzed with GraphPad Prism 7.0. For protein synthesis inhibition assays in HEK293T cells, cells were transiently transfected with the pNL3.2CMV (Promega) rather than by lentiviral transduction. Cells were plated in a 6-well plate at 1 × 106 cells/well and transfected the following day with 3.0 µg of plasmid DNA, in a 3:1 ratio of FuGENE®HD (Promega) transfection reagent to DNA. After 24 h, cells were re-plated at 5 × 103 cells/well, in a white clear-bottom 96-well plate and treated the following day with toxin. After overnight incubation (20 h), luminescence signal was developed using the NanoGlo® Luciferase Assay kit (Promega) similar to above. Each construct was tested 3 times with 2 technical replicates (at a minimum). Wildtype and DPH4-/- HEK293T cells were a gift from Dr. Mikko Taipale at the University of Toronto.
Nano glo luciferase assay kit
Manufactured by Promega
Sourced in United States
The Nano-Glo luciferase assay kit is a bioluminescent reporter assay system used to detect and quantify the activity of luciferase enzymes. The kit contains the necessary reagents to perform luminescence-based detection and measurement of luciferase activity in various experimental settings.
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Lab products found in correlation
Blood collection tube, by BD (4 mentions)
Cytation 3 cell imaging multi mode reader, by Agilent Technologies (3 mentions)
Fugene hd transfection reagent, by Promega (3 mentions)
Glomax multi detection system, by Promega (2 mentions)
3 mm glass beads, by Thermo Fisher Scientific (2 mentions)
Passive lysis buffer (plb), by Promega (2 mentions)
Glomax luminometer, by Promega (2 mentions)
Dual luciferase assay kit, by Promega (2 mentions)
Mouse d dimer elisa kit, by Elabscience (2 mentions)
Flexstation 3 instrument, by Molecular Devices (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Bioruptor sonicator, by Cosmo Bio (1 mentions)
Optiplate 96, by PerkinElmer (1 mentions)
White 96 well plate, by Greiner (1 mentions)
Purexpress δ ribosome kit, by New England Biolabs (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Passive lysate buffer, by Promega (1 mentions)
Ivis spectrum ct imaging system, by PerkinElmer (1 mentions)
Luciferase reagent, by Promega (1 mentions)
Fluoroskan ascent fl, by Thermo Fisher Scientific (1 mentions)
40 protocols using nano glo luciferase assay kit
1
Measuring Protein Synthesis Inhibition
2
Fecal Luciferase Quantification in Mice
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Fecal samples collected from Ifngr1−/− and NSG mice were analyzed with the Promega Nano-Glo luciferase assay kit as previously described [48 (link)]. Tubes for fecal collection were weighed before and after collection to determine fecal sample weight. Glass beads were added to the sample along with a fecal lysis buffer and vortexed thoroughly. Samples were then centrifuged, supernatant was extracted, and mixed with the Promega Nano-Glo luciferase assay kit buffer and substrate. Luciferase values were then read on the Cytation 3 cell imaging multi-mode reader, and the number of relative luminescence units per milligram of feces was calculated by dividing the average values of two technical replicates by the weight of the fecal sample.
3
Hydrodynamic Injection and APAP Assay
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Two milliliters (10% of the body weight in grams) of Ringer’s solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl2) containing 30 µg pTS864 (PhCMV-DARPinB4-EpoR-pA), 30 μg pTS922 (PhCMV-scFv-EpoR-pA) and 300 μg pJH6 (PSTAT3-NanoLuc-pA::PhCMV-STAT3-pA) was hydrodynamically injected into mice via a tail vein within 3–5 s. After the injection, mice were immediately fasted and randomly divided into two groups. Fifteen hours after plasmid injection, fasted mice were intraperitoneally injected with APAP (0 or 160 mg kg−1, Absin, cat. no. abs815915–500mg). Blood AST, ALT and Nluc levels were quantified at 8 h and 12 h post intraperitoneal APAP injection. Eight and twelve hours after APAP injection, blood samples were taken, allowed to clot, and centrifuged (2,700g for 10 min) in blood collection tubes (BD, cat. no. 365974) for analytical assays. Plasma AST and ALT levels were measured using an AST detection kit (KHB; SHFDA: 20142400181) and an ALT detection kit (KHB; SHFDA: 20172400610) according to the manufacturer’s instruction. Plasma Nluc levels were measured using a Nano-Glo Luciferase Assay kit (Promega, cat. no. N1120) according to the manufacturer’s instruction.
4
Quantifying Transient Gene Expression in Plant Protoplasts
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107 protoplasts were co-transformed with 3 µg of the NanoLuc vector and 3 µg of the GFP vector pCop007. Transformed protoplasts were incubated at 28 °C for 16–24 hrs. After incubation, protoplasts were centrifuged for 5 min at 600 g. Pellets were re-suspended in 100 µl of phosphate buffer (pH 5.5), mixed by pipetting, and disrupted by 10 time cycles of the 30 sec-sonication with the Bioruptor sonicator (CosmoBio) with HIGH setting/the 30 sec-cooling down on ice. The cell lysates were transferred to OptiPlate-96 (PerkinElmer), and the Luc activity quantified with a Nano-Glo Luciferase assay kit (Promega) following the manufacturer’s protocol. Luminescence was measured with a luminometer GloMax®-Multi Detection System (Promega). Normalized luminescence of each sample was calculated by dividing the luminescence by the transformation rate as following.
5
Nluc-based Parasite Invasion Assay
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Freshly egressed Nluc expressing parasites were harvested and passed through a 25-gauge needle 3 times to liberate from host cells. Parasites were counted and then preincubated for 20 minutes with different concentration of the compounds, ranging from 1,000 μM to 8 μM diluted in DMEM supplemented with 5% FBS. Parasites were then transferred into 96-well plates containing human foreskin fibroblasts in triplicate and centrifuged at 290g for 5 minutes at room temperature. Plates were then transferred to an incubator and the parasites were allowed to invade for 1 hour at 37°C in the presence of 10% CO2. Wells were then washed with DMEM 4 times to remove any noninvaded parasites. Plates were then placed at 37°C with 10% CO2 for 24 hours in 200 μL DMEM supplemented with 5% FBS. Invasion media was then removed and the host cells containing the Nluc expressing parasites were then lysed using Promega Nano-Glo luciferase assay kit and the light units quantified on a Millennium Science plate reader.
6
Luciferase Assays for Infected ALI and Mouse Fecal Samples
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All luciferase assays were performed with the Nano-Glo Luciferase Assay kit (Promega). For infected ALI cultures, transwells were incubated in 100 μl Nano-Glo Luciferase buffer (top compartment only) at 37°C for 15 mins. Cells were then scraped from the transwell membrane with a blunt pipette tip and transferred to a single well of a white 96-well plate (Greiner Bio-One). 100 μl of a 25:1 Nano-Glo Luciferase buffer to Nano-Glo Luciferase substrate mix was added to each well, and the plate was incubated for 3 min at room temperature. Luminescence values were read on a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek). For mouse fecal pellets in 1.7 ml microcentrifuge tubes, pellets were ground with a pestle, then 3-mm glass beads (Fisher Scientific) and 1 ml fecal lysis buffer (50 mM Tris pH7.6, 2 mM DTT, 2 mM EDTA pH 8.0, 10% glycerol, 1% Triton X-100 prepared in water) (Pawlowic et al., 2017 ) were added to the tube. Tubes were incubated at 4°C for 30 mins, vortexed for 1 min, then spun at 16,000 x g for 1 min to pellet debris. 100 μl supernatant was added to two wells of a white 96-well plate, then substrate addition and luminescence reading was performed as above.
7
SARS-CoV-2 Reverse Genetic Assay
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A total of 3 μg of plasmid DNA (containing icDNA of SARS-CoV-2) and 3 μl of Lipofectamine LTX with 3 μl of PLUS reagent were used to transfect BHK-21 cells (ECACC) in 6-well plates. On the next 3 days posttransfection, supernatant was transferred to Vero E6 cultures in T25 flasks. Virus was allowed to propagate in Vero E6 cells for a further 4 days before harvesting the P0 stock. Infectious titres were enumerated by counting infected foci or via plaque assay. Rescued virus was used to infect Vero E6 cells in 96-well plates. For the Nanoluc assay, cells were lysed using a passive lysis buffer (Promega, cat#E1941) at 24 hpi. NanoLuc activity was determined using the Nano-Glo Luciferase Assay kit (Promega, Cat# N1130) and measured using a GloMax Luminometer (Promega).
8
Luciferase Reporter Assay for Promoter Analysis
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For the luciferase reporter analysis, we modified the pHRP309 plasmid70 (link) and replaced the LacZ reporter gene with the Nluc reporter gene. Putative promoter regions were PCR-amplified and cloned upstream of the Nluc reporter gene. These plasmids were conjugated into the WT, ΔtagH, and ΔtagH::tagH strains, respectively. The strains were then cultivated in LB medium supplemented with 50 µg/mL gentamicin at 37°C with shaking at 220 rpm until the OD600 reached ~0.6. Cell pellets were collected, washed twice with ice-cold PBS, and resuspended in an equal volume of cold PBS. Cells were disrupted using ultrasound, and the resulting cell supernatants were detected using a Nano-Glo luciferase assay kit (Promega). All experiments were repeated three times for each group.
9
In vitro Translation of NanoLuc Luciferase
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In vitro translation of NanoLuc was performed using a PURExpress ΔRibosome kit (NEB) as previously described (11 (link)). Briefly, 2.0 μl of solution A, 0.6 μl of factor mix, and 18 ng of DNA template (containing the T7 promoter, a 5′ untranscribed region [UTR] with a Shine-Dalgarno sequence, and the NanoLuc coding sequence) were mixed with 12.5 nM purified 70S ribosomes in a total volume of 5 μl. After 1 h of incubation at 37°C, relative luminescence units (RLU) produced from the translated luciferase was measured with a Nano-Glo luciferase assay kit (Promega Inc.) according to the manufacturer’s instructions. The inhibition of translation activity of 70S ribosomes purified from designated strains was assayed by adding the designated antibiotic at specified concentrations in the reaction mixture prior to ribosome addition and measuring luciferase activity after 1 h of incubation at 37°C. A reaction mixture without the antibiotic was used as a reference to determine percent inhibition. The IC50 value from a dose-response curve was obtained from nonlinear regression of log10 inhibitor versus activity plot using the variable slope model of GraphPad Prism software. The 95% confidence interval for each best-fit curve, also computed by Graph-Prism software from the standard errors, represents 95% probability that the computed IC50 value is in the given range.
10
Generating ZIKV Pseudotyped Lentivirus for Cell Transduction
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ZIKV prME protein-pseudotyped MLV expressing nanoluciferase was generated as previously described [35 (link),38 (link)]. Briefly, HEK293T cells were transfected with pUMVC, pBABE-puro-NanoLuc, and a mammalian vector expressing the ZIKV CaprME. The culture supernatant harvested 2 days after transfection was centrifuged at 1500 × g for 10 min and passed through 0.45 μm filter to remove cell debris. Pseudotyped MLV titre was quantified using qRT-PCR. Packaged viral RNA was extracted using TRIzol LS reagent (Invitrogen) according to the manufacturer’s instructions and subjected to reverse transcription and qRT-PCR using primers targeting the 5′ LTR region of MLV RNA (forward primer 5′-ATTGACTGAGTCGCCCGG-3′ and reverse primer 5′-AGCGAGACCACAAGTCGGAT-3′). To transduce HEK293T cells with the pseudotyped virus, target cells were seeded in a 12-well cell culture plate and inoculated with 0.5 ml media containing pseudotyped virus for 9 h. Cells were then washed and further cultivated in fresh complete media for 48 h prior to measuring reporter activity using a Nano-Glo Luciferase assay kit (Promega) and the GloMax-Multi Detection System (Promega).
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