The luciferase reporter assay was performed as previously described11 (link). CD47 LightSwitch Promoter Reporter GoClones (RenSP, S710450) and Cypridina TK Control constructs (pTK-Cluc, SN0322S) were obtained from SwitchGear Genomics. 45 ng of the RenSP reporter and 5 ng of the pTK-Cluc reporter construct were transfected into mouse smooth muscle cells using Lipofectamine 3000 Transfection Reagent (Thermo Scientific, Catalogue# L3000–008) and Opti-MEM I Reduced Serum Medium (Thermo Scientific, Catalogue# 31985062). After 48 hours, media was changed to fresh medium and cells were then exposed to DMSO, 50 ng/ml TNF-α + DMSO, or 50 ng/ml TNF-α + 10 μM atorvastatin. The cell lysate and supernatant were harvested 24 hours after stimulation/treatment und dual luciferase activity was measured with the LightSwitch Luciferase Assay Kit (Active Motif, Catalogue# 32031, NC0999256) and Pierce Cypridina Luciferase Glow Assay Kit (Thermo Scientific, PI16170) using an iD3 luminometer (Molecular Devices). Relative luciferase activity (RenSP/Cypridina ratio) was quantified as the percentage change relative to the basal value obtained from control-transfected cells.
Pierce cypridina luciferase glow assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce Cypridina Luciferase Glow Assay Kit is a laboratory tool used to measure the activity of the Cypridina luciferase enzyme. The kit provides the necessary reagents to perform a luminescent-based assay for quantifying the Cypridina luciferase present in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lightswitch luciferase assay kit, by Active Motif (2 mentions)
Id3 luminometer, by Molecular Devices (2 mentions)
Cd47 lightswitch promoter reporter goclones rensp, by SwitchGear Genomics (2 mentions)
Cypridina tk control, by SwitchGear Genomics (2 mentions)
Ptk cluc, by SwitchGear Genomics (2 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (2 mentions)
Pmcs cypridina luc, by Thermo Fisher Scientific (2 mentions)
Spectramax l luminometer, by Molecular Devices (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Synergy h1 hybrid, by Agilent Technologies (1 mentions)
Renilla glo luciferase assay system, by Promega (1 mentions)
Pierce gaussia luciferase flash assay kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using pierce cypridina luciferase glow assay kit
1
CD47 Promoter Reporter Assay
2
CD47 Promoter Reporter Assay
3
Cypridina Luciferase Reporter Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
We used secreted luciferase from Cypridina. The Pgk1 promoter (−648 to −50) was amplified from mouse genomic DNA by PCR and cloned into the Cypridina luciferase reporter plasmid pMCS-Cypridina Luc (Thermo Fisher Scientific) at the XhoI and BamHI restriction sites. sPLA2-V-WT and sPLA2-V KD cells as well as sPLA2-V KD cells expressing sPLA2-V-H48Q were transfected with Cypridina luciferase reporter vector using FuGENE HD Transfection Reagent (Promega) according to the manufacturer’s instructions. Renilla luciferase expression vector (pTK-Green Renilla Luc, Thermo Fisher Scientific) was co-transfected to serve as an internal control. At 24 hr post transfection, the culture medium and the cells were harvested. Cypridina luciferase activity in the harvested culture medium was measured using a SpectraMax L luminometer (Molecular Devices, San Jose, CA, USA) and a Pierce Cypridina Luciferase Glow Assay Kit (Thermo Fisher Scientific). Renilla luciferase activity in cellular lysates was measured on the luminometer using coelenterazine as a substrate. Cypridina luciferase activity was normalized to Renilla luciferase activity. Data are represented as fold-induction by normalizing the luciferase activity of the tested sample to that of the corresponding control sample.
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We used secreted luciferase from Cypridina. The Pgk1 promoter (−648 to −50) was amplified from mouse genomic DNA by PCR and cloned into the Cypridina luciferase reporter plasmid pMCS-Cypridina Luc (Thermo Fisher Scientific) at the XhoI and BamHI restriction sites. sPLA2-V-WT and sPLA2-V KD cells as well as sPLA2-V KD cells expressing sPLA2-V-H48Q were transfected with Cypridina luciferase reporter vector using FuGENE HD Transfection Reagent (Promega) according to the manufacturer’s instructions. Renilla luciferase expression vector (pTK-Green Renilla Luc, Thermo Fisher Scientific) was co-transfected to serve as an internal control. At 24 hr post transfection, the culture medium and the cells were harvested. Cypridina luciferase activity in the harvested culture medium was measured using a SpectraMax L luminometer (Molecular Devices, San Jose, CA, USA) and a Pierce Cypridina Luciferase Glow Assay Kit (Thermo Fisher Scientific). Renilla luciferase activity in cellular lysates was measured on the luminometer using coelenterazine as a substrate. Cypridina luciferase activity was normalized to Renilla luciferase activity. Data are represented as fold-induction by normalizing the luciferase activity of the tested sample to that of the corresponding control sample.
4
SLC30A2 Promoter Luciferase Assay
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SLC30A2 promoter‐Cypridina luciferase reporter plasmids, in which WT or mutant SLC30A2 promoter (from −363 to −32 with reference to the adenine base of the start codon methionine as +1) is inserted into the multiple cloning site of pMCS‐Cypridina Luc (Thermo Scientific, Waltham, MA, USA), were transiently transfected into A549, MCF7, or MDA‐MB‐231 cells, as described previously;28 Renilla luciferase expression plasmid was simultaneously transfected. The activities of Cypridina and Renilla luciferase were measured using Pierce Cypridina Luciferase Glow Assay Kit (Thermo Fisher Scientific, Rockford, IL) and Renilla‐Glo Luciferase Assay System (Promega, Madison, WI, USA), respectively, using a Synergy H1 Hybrid multimode microplate reader (BioTek, Winooski, VT, USA). Cypridina luciferase activity was divided by that of Renilla luciferase for normalization of transfection efficiency.
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SLC30A2 promoter‐Cypridina luciferase reporter plasmids, in which WT or mutant SLC30A2 promoter (from −363 to −32 with reference to the adenine base of the start codon methionine as +1) is inserted into the multiple cloning site of pMCS‐Cypridina Luc (Thermo Scientific, Waltham, MA, USA), were transiently transfected into A549, MCF7, or MDA‐MB‐231 cells, as described previously;
5
Luciferase Assay for Nonsense Mutation Readthrough
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