TNFα-Mediated Activation of NF-kB
One of the most important downstream signaling targets activated by TNFα is the NF-kB transcription factor, which is involved in diverse biological processes including inflammatory, antiapoptotic, and immune responses. In resting cells, the activity of NF-kB is controlled through its cytoplasmic sequestration by inhibitor of kappa B (IkB). Binding of TNFα to its cell surface receptors initiates a series of kinase cascades, which lead to phosphorylation of IkB, ubiquitination, and proteasome-dependent degradation. The released NF-kB then undergoes nuclear translocation and activation, which induces the transcriptional expression of target genes (Figure 1).
Luciferase reporter assays have been used widely to investigate cellular signaling pathways. In this study, the GloResponse NF-kB-RE-luc2P HEK293 cell line from Promega was used. This line, a clonal derivative of HEK293 cells, contains a luciferase gene (luc2P) under the control of a minimal TATA promoter with multiple Nuclear Factor-kB Response Elements (NF-kB-REs). This NF-kB reporter cell line is designed for rapid analysis of any cellular response that results in modulation of NF-kB activities.
To evaluate TNFα-mediated activation of NF-kB, GloResponse NF-kB-RE-luc2P HEK293 cells were robotically dispensed using Eppendorf’s epMotion 5075 robotic liquid-handling workstation at a density of 10,000 cells per well into white-bottom, white-opaque 96-well Costar #3917 microplates from Corning in 50 µL volumes of DMEM supplemented with 10% fetal bovine serum (FBS).
Threefold serial dilution treatments of TNFα in 50 µL media per well were robotically dispensed across the plate. Treated cells were allowed to incubate for five hours at 37ºC, 5% CO2. Controls were included on each plate: one column of wells containing untreated cells and one cell-free column.
To evaluate anti-TNFα-mediated neutralization of NF-kB, GloResponse NF-kB-RE-luc2P HEK293 cells were dispensed as described previously using Tecan’s Freedom EVO robotic workstation and treated immediately with 10 ng/mL of TNFα and increasing concentrations of antihuman TNFα mAb from R&D Systems. Threefold serial dilutions of the anti-TNFα mAb plus TNFα in 50 µL media per well were robotically dispensed across the plate. Treated cells were allowed to incubate for five hours at 37ºC, 5% CO2. Controls were also included on the plate.
Following treatment, 100 µL of the ONE-Glo reagent was robotically added to each well of all plates and contents were mixed on a rotating platform for 10 minutes. Luminescence associated with luciferase gene expression due to modulation of NF-kB activities was measured using Promega’s GloMax-Multi Plate Reader.
Z´-factor analysis, a statistical measure of reproducibility and robustness, was also performed for each assay. A concentration of 10 ng/mL TNFα or 10 ng/mL TNFα plus 1 µg/mL anti-TNFα in 50 µL media per well was used for induction in part of the plate, whereas the other portion of the plate was left untreated. Cells were robotically prepared and treated similarly to the previously described methods.
Dose-response curve results using the ONE-Glo Assay are shown in Figure 2 for TNFα- and anti-TNFα-treated GloResponse NF-kB-RE-luc2P HEK293 cells dispensed robotically in 96-well plates. The EC50 value, or half maximal effective concentration of TNFα induction, obtained for suspension cell conditions with TNFα-treatment alone was 1.1 ng/mL.
The ND50 value, or half neutralization dose, obtained for suspension-cell conditions for TNFα neutralization was 78 ng/mL. Z´-factor values for both automated treatment conditions were >0.71, indicating excellent assay quality. In addition, both treatment conditions showed a great dynamic response.