Dual-Luciferase Reporter Assay for Detection of NF-κB Activity

Transient transfection followed by luciferase assay was performed to determine the NF-κB activity rate. Cells were seeded in 96-wells microplate and incubated O/N to reach greater than 50% confluency. Co-transfection of the plasmids NF-κB-RE Firefly luciferase reporter vector and Renilla as control vector was performed using FuGENE HD transfection reagent (Promega, Madison, WI, USA) accordingly to the manufacturer's instructions. Cells were incubated at 37°C and allowed to recover for 24 hours before administering treatments (2 hours of incubation with L.SK extract). The expression of luciferase enzyme activated by NF-κB binding was determined using the Dual-Luciferase Reporter Assay System (Promega), by following the manufacturer's protocol. The results of NF-κB activity were expressed as the relative promoter-luciferase activity, normalizing for Renilla activity.