Cell extracts were prepared and luciferase activity was measured using the One-Glo Luciferase Reporter Assay System, according to the manufacturer's instructions (Promega Corporation, Madison, WI, USA). The luciferase activity assays were performed with a 20/20 luminometer (Turner BioSystems, Sunnyvale, CA, USA). To investigate the effect of CD271 blocking antibodies on ethanol-induced nuclear factor-κB (NF-κB) activation, cells were pre-incubated with 10 µg/ml monoclonal mouse anti-human CD271 antibody [clone ME20.4 (catalog no. 345102; BioLegend, Inc., San Diego, CA, USA) or clone C40-1457 (BD Biosciences)] for 1 h prior to addition of ethanol.
One glo luciferase reporter assay system
Manufactured by Promega
Sourced in United States
The One-Glo Luciferase Reporter Assay System is a luminescence-based assay that measures the activity of luciferase reporter genes in mammalian cells. The system provides a sensitive and quantitative method for monitoring gene expression.
Automatically generated - may contain errors
Lab products found in correlation
C40 1457, by BD (1 mentions)
Luminescence plate reader, by Berthold Technologies (1 mentions)
Lipofectamine transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
5 protocols using one glo luciferase reporter assay system
1
Ethanol-Induced NF-κB Activation and CD271 Antibody
2
Luciferase Assay for PPARα Activity
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For luciferase assays, HepG2 cells were seeded at a density of 1 × 104 cells in a 96-well plate. Cells were transfected with lipofectamine transfection reagent (Thermo Fisher Scientific, Rockford, IL, USA), and plasmids were used for transfection with the PPRE-X3-TK-LUC plasmid (Dr. Christoper K. Glass, University of California, San Diego, CA, USA) and PPARα expression vectors (Dr. Han Geuk Seo, Konkuk University, Seoul, South Korea). After transfection for 24 h, cells were treated with WY14643 (a PPARα agonist) [71 (link)] or SSC for 6 h. Luciferase activity was measured using the One-Glo Luciferase Reporter Assay System (Promega, Madison, WI, USA) and a luminescence plate reader (Berthold Technologies GmbH & Co., Bad Wildbad, Germany.
3
NF-κB Activity Modulation in HaCaT Cells
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HaCaT cells were transfected with 0.1 µg pGL4.32 (luc2P/NF-κB-RE/Hygro) plasmids (Promega Corporation), using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. At 24 h after transfection, the cells were pretreated with COEE (2.5, 5, 10 and 20 µg/ml) and Bay11-7082 (5 µM) for 1 h at 37°C, stimulated with TNF-α/IFN-γ for 20 h at 37°C, harvested and then assessed for luc2P luciferase activity using the ONE-Glo™ luciferase reporter assay system (Promega Corporation) according to the manufacturer's instructions. Normalization was performed by comparison with Renilla luciferase activity.
4
Pseudomonas aeruginosa-Induced NF-κB Activation
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Nasal fibroblasts were transfected in A549 cells with pGL4.43 (luc2P/NF-κB-RE/Hygro) plasmid using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's protocol. We used the human lung epithelial cell line A549, purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Twenty hours after transfection, cells were stimulated with P. aeruginosa strain K for 2 h, harvested, and then assessed for luciferase activity using the ONE-Glo luciferase reporter assay system (Promega, Madison, WI) according to the manufacturer's instructions.
5
Transient HNF1B Expression Assay
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Human embryonic kidney 293 cells were seeded in 96-well plates (approximately 70–80%
confluence) and transiently transfected with 100 ng of the 4×HNF-luc reporter vector
together with 3 ng of each HNF1B expression vector (WT or Leu168Pro) or an empty vector.
Firefly luciferase activity was measured 48 h after transfection using ONE-Glo Luciferase
Reporter Assay System (Promega) and a FlexStation 3 microplate reader (Molecular Devices,
San Jose, CA, USA). Luciferase activity was measured in triplicate, and all assays were
repeated at least thrice. Statistical significance was determined using the t-test. A
two-tailed P-value with an alpha level for significance was set at ≤
0.05.
confluence) and transiently transfected with 100 ng of the 4×HNF-luc reporter vector
together with 3 ng of each HNF1B expression vector (WT or Leu168Pro) or an empty vector.
Firefly luciferase activity was measured 48 h after transfection using ONE-Glo Luciferase
Reporter Assay System (Promega) and a FlexStation 3 microplate reader (Molecular Devices,
San Jose, CA, USA). Luciferase activity was measured in triplicate, and all assays were
repeated at least thrice. Statistical significance was determined using the t-test. A
two-tailed P-value with an alpha level for significance was set at ≤
0.05.
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