The abundance of Nluc was measured using the Nano-Glo Luciferase system (Promega, N1110). For this experiment, 3 ml cultures of Mfr were grown in the previously described conditions until exponential phase. At this point, 1 ml of each culture was taken and centrifuged at 10,000 rpm. The supernatants were kept on ice until testing. The pellets were washed three times and finally resuspended in 1 ml PBS 1x. In both the supernatants and the pellets, 50 µl of samples were mixed with a 50 µl of a substrate + buffer mix (1:50 ratio) in a flat white 96-well plate (Corning, CLS3917). The plates were incubated in the dark for 10 min prior to reading the luminescence at 1000 ms integration time, 50 ms settle time in an Infinite 200 Pro plate reader (Tecan). Luminescence signal (arbitrary units, a.u.) was normalised by measuring the protein concentration in each sample. For this, 200 µl of the pellet resuspension was centrifuged again and resuspended in SDS 1% lysis buffer. They were sonicated using a Bioruptor sonication system (Diagenode) and On/Off cycles of 30 s. each for 10 min., centrifuged and the protein concentration was determined with the Pierce BCA Protein Assay kit.
Nano glo luciferase system
Manufactured by Promega
Sourced in Germany, United States
The Nano-Glo luciferase system is a bioluminescent reporter technology developed by Promega. It utilizes a luciferase enzyme that catalyzes a light-emitting reaction upon the addition of a substrate. The system is designed to provide a sensitive and quantitative measurement of gene expression or other cellular events.
Automatically generated - may contain errors
Lab products found in correlation
Biostack4, by Agilent Technologies (2 mentions)
Infinite 200 pro plate reader, by Tecan (1 mentions)
Bioruptor sonication system, by Diagenode (1 mentions)
Orion microplate luminometer, by Berthold Technologies (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
Peg virus precipitation kit, by Abcam (1 mentions)
0.45 μm filter, by Merck Group (1 mentions)
Steady glo luciferase system, by Promega (1 mentions)
Steady glo luciferase assay system, by Promega (1 mentions)
8 protocols using nano glo luciferase system
1
Quantifying Nluc Abundance in Mfr
2
Nano-Glo Luciferase Assay Protocol
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nLuc assays were using the Nano-Glo Luciferase system (Promega) and preformed according to manufacturer’s guidelines. Samples were diluted in 1x PLB (25 μl total volume) for determination of nLuc relative light units (RLU) using a Orion microplate luminometer (Berthold) or a FLUOstar Omega microplate reader (BMG Labtech). RLU was normalized to protein levels in samples determined by a bicinchoninic acid protein assay (Pierce).
3
Pseudovirus Infectivity Assay in HEK 293T-hACE2 Cells
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For pseudovirus infectivity experiments, HEK 293T-hACE2 cells (2.5 × 104 cells/well) were seeded in a 96-well plate. Cells were infected the next day and lysed at 48 hpi. Luminescence was measured using Nano-Glo luciferase system (Promega) and a Biostack4 (BioTek) luminometer. Infectivity was determined by normalizing the luciferase signals to virus levels as determined by Western blots probing for HIV-1 p24CA on culture supernatants.
4
Neutralizing Antibody Assay for CHIKV and SFV
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Assessment of neutralizing antibodies in mice was performed using a modified luciferase based assay (described in [31 (link)]). CHIKV and SFV clones expressing NanoLuc luciferase (nLuc) (Promega, Madison, WI) in-frame between the capsid and E3 proteins were engineered as previously described [32 (link)]. Neutralization was performed by incubating heat-inactivated serum diluted 1:20 with 5x103 PFU of either CHIKV or SFV expressing nLuc overnight at 4°C. Confluent BHK-21 in 96 well plates were then infected in triplicate with serum: virus mixture for one hour, followed by washing and addition of fresh media. Five hours post-infection, media was discarded and cells were lysed and analyzed for luciferase expression using the Nano-Glo luciferase system (Promega). Data are expressed as fold-neutralization using normal mouse serum for normalization. Plaque reduction neutralization 50% (PRNT50) assay in BHK-21 cells was used for determination of neutralizing titers in human samples (described in [33 (link)]).
5
Production and Infection of Hepatitis B Virus
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Wild-type HBV was derived from the supernatants of HepG2.2.15 cells, which were stably transfected with a complete HBV genome. HBV reporter viruses (HBV-NL) were produced by transient transfection of HepG2 cells with pUC1.2-HBV/NL and pUC-HBV-D, as previously described [21 (link)]. The collected supernatants were filtered through a 0.45-μm filter (Merck Millipore), and concentrated approximately 100 times using a PEG Virus Precipitation kit (BioVision). Cells were infected with wild-type HBV at a concentration of 5,000 genome equivalents per cell in the presence of 4% PEG8000 for 16 hours. Alternatively, cells in a 96-well plate were inoculated with 5 μl of HBV-NL in the presence of 4% PEG8000 for 16 hours. HBV-infected cells were cultured in fresh medium for an additional 5–6 days and their infectivity was determined by intracellular HBcAg staining or extracellular HBsAg quantification, as previously described [28 (link)]. The infectivity of HBV-NL was quantified using the Nano-Glo Luciferase System (Promega), according to the manufacturer's instructions.
6
Nanoluciferase-based Viral Entry Assay
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A total of 10 × 104 CrFK cells or 5 × 103 U-87 MG cells were seeded into 96-well plates, and infection was performed the following day. For experiments involving cyclosporin A (CsA, Sigma-Aldrich, Germany), 1 to 10 µM of CsA or control DMSO was used to treat cells 2 h before infection. Cells were then infected with different reporter viral particles, and after 48 to 72 h, luciferase activity was measured. For infection with nanoluciferase-containing reporter viruses, cells were washed with phosphate-buffered saline three times before lysis and luciferase measurement, in addition to medium change 24 h following infection, to eliminate the effect of background nanoluciferase. Nanoluciferase activity was measured with the Nano-Glo Luciferase system (Promega, Mannheim, Germany), and firefly luciferase activity was measured with the Steady-Glo Luciferase system (Promega) on a MicroLumat Plus luminometer (Berthold Detection Systems, Pforzheim, Germany). Each experiment was performed in triplicates for at least three times.
7
Pseudovirion Transduction Assay in Calu-3 Cells
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Calu-3 cells (10,000 cells/well plated in a 96-well dish for 48 h) were transduced with 12 ng p24 equivalent of filtered pseudovirions overnight [9 (link)]. Target cells were pretreated with BOS-981 (1 μM) for 6 h before transduction. The overnight incubation with pseudovirions was performed in the presence of the inhibitors. Viral inoculum was removed, then fresh media were added, and the cells were cultured for up to 72 h. Upon removal of spent media, Calu-3 cells were gently washed twice with PBS and analyzed for nanoluciferase activity, respectively, using the Promega Nano-Glo luciferase system (Madison, WI, USA).
8
Pseudotyped SARS-CoV-2 Spike Protein Infectivity
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For infections using the SARS-CoV-2 S MLV pseudotypes, 293T-hACE2 cells (2.5 × 104 cells/well) were seeded in a 24-well plate. Cells were infected the following day and lysed 48 hpi followed by measuring luminescence using the Steady-Glo luciferase assay system (Promega) per manufacturer’s recommendation and Biostack4 (BioTek) luminometer, an automated plate reader. For SARS-CoV-2 S/CoV S HIV pseudotypes, Calu-3 or 293T-hACE2 cells (2.5 × 104 cells/well) were seeded in a 96-well plate. Cells were infected the next day and lysed 48 hpi followed by measuring luminescence using the Nano-Glo luciferase system (Promega) per manufacturer’s recommendation and a Biostack4 (BioTek) luminometer. Infectivity was determined by normalizing the luciferase signals to virus levels as determined by western blots probing for MLV p30CA or HIV p24CA on culture supernatants (see Immunoblotting section).
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