Measurement of NF-κB Activity in Caco-2 Cells

An NF-κB luciferase reporter plasmid (500 ng; Ehrentraut et al., 2013 (link)) and a Renilla reporter plasmid (1 ng) were transfected into subconfluent Caco-2 cells using Lipofectamine LTX Reagent (Thermo Scientific). Luciferase activity was determined at 16 h using Promega dual luciferase reagents (Promega, Madison, WI), and luminescence was determined using the GloMax-Multi plate reader (Promega). Each experiment was performed in triplicate.

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Ehrentraut S.F., Curtis V.F., Wang R.X., Saeedi B.J., Ehrentraut H., Onyiah J.C., Kelly C.J., Campbell E.L., Glover L.E., Kominsky D.J, & Colgan S.P. (2016). Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation. Molecular Biology of the Cell, 27(23), 3687-3694.

Publication 2016

Lipofectamine LTX Reagent GloMax-Multi plate reader

Caco 2 cells Lipofectamine Luciferase Luminescence Nf κb Plasmid Promega Renilla

Corresponding Organization : VA Eastern Colorado Health Care System

Other organizations : University Hospital Bonn, Montana State University

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Variable analysis

independent variables

Transfection of NF-κB luciferase reporter plasmid (500 ng) and Renilla reporter plasmid (1 ng) into Caco-2 cells

dependent variables

Luciferase activity determined at 16 h using Promega dual luciferase reagents

control variables

Caco-2 cell line
Lipofectamine LTX Reagent (Thermo Scientific) used for transfection
Experiments performed in triplicate

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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