An IL-10RA-luciferase reporter plasmid and empty vector control (Switchgear Genomics) were transfected into Caco-2 cells using Lipofectamine 3000 transfection reagent (Invitrogen) using the manufacturer’s recommended protocol. The day following transfection, cells were treated with the designated dose of butyrate. Cells were lysed the day after treatment and luciferase (Promega) was measured and normalized to protein by BCA. Lightswitch (Active Motif) were used to quantify reporters from Switchgear Genomics.
Lightswitch
Manufactured by Active Motif
Sourced in United States
The Lightswitch is a laboratory instrument designed for the detection and quantification of luciferase reporter gene activity. It functions by measuring the luminescence generated from the enzymatic conversion of a substrate, providing a reliable and sensitive method for assessing gene expression levels.
Automatically generated - may contain errors
3 protocols using lightswitch
1
Butyrate Modulates IL-10RA Expression
2
Luciferase Reporter Activity Assay
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Luciferase reporter activity assay was performed, as described previously, by transfecting the cells with the LightSwitch™ luciferase reporter gene vector under the influence of the MR promoter (Active Motif, Inc., Carlsbad, CA, United States)[11 ]. The measurements were done the next day with the manufacturer’s assay kit and according to the manufacturer’s instructions.
3
Investigating VEGF-C Promoter Regulation
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CAL33 cells belonging to the CR-MI group were transfected by using 50 μl NaCl buffer, 1.25 μl of polyethylenimine transfection reagent (Sigma Aldrich) and 0.5 μg of total test plasmid DNA-renilla luciferase. The plasmids encoded either (i) a human vegf-c promoter fragment with either a non-mutated (wild type, WT) or a mutated (MUT) binding site for the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB),32 (link) (ii) an artificial promoter containing three binding sites for human NF-κB or (iii) a human VEGF-C 3′UTR reporter (LightSwitch, S803537, Active Motif, Carlsbad, CA, USA), all cloned downstream of the luciferase reporter gene. A CMV plasmid was used to control the variability of transfection efficiency in the reporter assays.
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