NFκB-Luciferase Reporter Assay

Keratinocytes were seeded into 12-well plate at a density of 150,000 cells/ml in keratinocyte serum-free medium. 24 h later cells were transfected with the pNFκB-luc Cis-Reporter Plasmid (Stratagene) reporter construct vector and the pGL4.75 [hRluc/CMV] plasmid (Promega) internal control using the X-tremeGeneHP transfection reagent (Roche Diagnostics). 24 h after transfection, cells were washed in PBS and lysed in passive lysis buffer (Biotium, Hayward, CA, USA). Luciferase activity of the lysates was measured using the Firefly & Renilla Dual Luciferase Assay Kit (Biotium) and Thermo Luminoskan Ascent software (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. Luciferase activity derived from NFκB-luc plasmid was normalized to the activity of Renilla luciferase activity from the pGL4.75 [hRluc/CMV] plasmid. The transfection efficiency was similar in the PRP patient and healthy-control-derived keratinocytes, as determined by the transfection of a GFP reporter construct (Lonza, Basel, Switzerland) and flow cytometry.