NF-κB reporter luciferase assays

HEk293T or Jurkat cells were transfected with plasmid DNAs encoding for NF-κB promoter-driven firefly luciferase (pNF-κB-Luc), and a Renilla luciferase (pRL-Tk) as the control. Jurkat T cells were transfected by electroporation using NeonTM transfection system (Invitrogen). 2 × 10⁶ cells in a total volume of 100 μl of serum free medium with the indicated plasmid DNA were electroporated using the following parameters (pulse voltage: 1325V, pulse width: 10 ms, pulse number: 3). HEk293T cells were transfected by the calcium phosphate-DNA precipitation method. 36 hrafter tansfection, the cells were lysed in 30 μl passive lysis buffer and the bioluminescence of the samples was measured using the Dual-Luciferase Reporter Assay System (Promega) and a Berthold Microplate Luminometer (Titertek Berthold). The relative luciferase activities were calculated after normalization of firefly luciferase activities to the activities of Renilla luciferase.