Hek dual tnfα cells

HEK-Dual TNFα cells (Invivogen, San Diego, CA; catalog #hkd-tnfa) were used to assess the ability of LMWF5A to inhibit NF-κB. These cells contain a reporter construct that encodes a secreted luciferase gene (Lucia) under the control of a NF-κB inducible promoter. 130 µL LMWF5A was added in triplicate to a 96-well plate. 70 µL HEK-Dual TNFα cells in DMEM (Corning, Manassas, VA) supplemented with 10% heat-inactivated FBS, 10 U/mL pencillin-100 µg/mL streptomycin, 100 µg/ml normocin (Invivogen), and 100 µg/ml zeocin (Invivogen) were then added for a final concentration of 50,000 cells/well, and the cells were incubated at 37 °C and 5% CO₂ for 24 h. To induce luciferase expression, the cells were stimulated with 2 ng/mL TNFα (Invivogen) for an additional 24 h at 37 °C and 5% CO₂. The luciferase activity was quantitated by combining 20 µL each cell supernatant and 100 µL QUANTI-Luc reagent (Invivogen) in a new, opaque 96-well plate and immediately measuring luminescence on a SpectraMax M5e and Flexstation 3 System (Molecular Devices, San Jose, CA).

Hek-Dual TNFα cells (Invivogen) derived from the human embryonic kidney 293 cell line by stable co-transfection of two NF-kB-inducible reporter constructs: the SEAP (secreted embryonic alkaline phosphatase) gene and the Lucia luciferase gene were used. Both reporter genes are under the control of the same NF-KB-inducible promoter. In this system, the cells specifically respond to TNF and not to other TLR/IL1 signals since they lack intracellular MyD88. Hek-Dual TNFα cells were stimulated over night with rTNFα (4 ng/ml, Peprotech) with or without the addition of amniotic fluid (AF) (dilution 1:10) obtained at delivery from Control (Ctrl) or Adalimumab injected animals. The presence or inhibition of bioactive TNFα was demonstrated by activation/inhibition of the AP-1/NF-KB pathway using QUANTI-Blue Solution (Invivogen) resulting in luciferase bio-luminiscence.