Comparison of Dual-Luciferase® Reporter Assay and a Stably Transfected NF-κB Reporter Cell Line for Detecting TNF-α and X-Ray-Induced Pathway Activation

The transcription factor nuclear factor kappa B (NF-κB) is a central regulator of inflammatory and genotoxic stress responses and is involved in immune function and disease pathogenesis. Reporter assays are widely used to quantify NF-κB activation, yet systematic benchmarks between transient dual-luciferase and stable fluorescent formats under harmonized conditions are lacking. We compared a transient Dual-Luciferase® Reporter (DLR™) assay in HEK293 cells with the stably transfected HEK-pNF-κB-d2EGFP/Neo L2 cell line using recombinant TNF-α and X-rays as stimuli. Dynamic range, limit of detection (LOD), signal-to-noise ratio (S/N), and half-maximal effective concentrations and doses (EC50, ED50) were derived from four-parameter logistic modeling. The HEK-pNF-κB-d2EGFP/Neo L2 line showed higher sensitivity to TNF-α (EC50 = 0.31 ng/mL) than the DLR™ assay (1.2–2.0 ng/mL) and a lower LOD, whereas the DLR™ assay provided better temporal resolution, revealing assay-specific signal decline attributable to reporter stability and readout kinetics. For X-ray exposure, both systems showed comparable potency (EC50 ≈ 5–8 Gy), while the stable cell line maintained superior S/N. Both reporter systems support high-throughput and mechanistic applications: the DLR™ assay offers versatility across cell types, while the HEK-pNF-κB-d2EGFP/Neo L2 line provides higher sensitivity, dynamic range, and single-cell resolution.