Recombinant murine tnf

Calvarial osteoblasts were isolated from TOPGAL transgenic mice (Jackson) that express a β-galactosidase reporter construct responsive to activation of canonical Wnt signaling, such that expression correlates with Wnt activity, as determined by the mammalian β-galactosidase assay kit (Thermo Scientific) with absorbance measurement at 405 nm. Cells were seeded in 96-well plates at 5000 cells/well. At 80 % confluency (day 0), differentiation media was added with 7.5 ng/ml recombinant murine TNF (R&D Systems) or 50 ng/ml IL-17A. β-galactosidase activity was determined on days 0, 7, 14, 21, and 28 of differentiation.

Polyclonal IgG to TNF (R&D systems) was diluted to 2 μg/ml in carbonate buffer and dispensed in 96-well Maxisorb Immunoplates overnight. Samples were added for one hour, followed by the detection antibody, a biotinylated anti-mouse TNF polyclonal antibody (R&D systems) added at 200 ng/ml. A 1:5,000 dilution of extravidin-alkaline phosphatase was then added for a further hour followed by the addition of 1 mg/ml 4-nitrophenyl phosphate. Plates were read at 450 nM and results were calculated by reference to a standard curve of recombinant murine TNF (R&D systems).

All experiments were performed in accordance with the institutional regulations. Murine hepatoma cell lines Hepa1-6 and Hep56 (CLS, Eppelheim, Germany) were seeded at a density of 4.0 × 10⁵ cells per 6 cm² dish (TPP, Trasadingen, Switzerland) and cultivated at 37°C in DMEM-medium +10% FCS and +1% penicillin/streptomycin (Sigma Aldrich, Taufkirchen, Germany) for 24 h. Three hours prior to TNF stimulation, medium was removed, cells were washed with DMEM-medium and further cultivated in medium without FCS. For single stimulation of cells, 10 ng/ml recombinant murine TNF (R&D Systems, Minneapolis, USA) was used. For triple stimulations, 1 ng, 2.5 ng, and 5 ng/ml (time points 0, 60, and 120 min) were added to the medium. Medium was removed after 5, 10, 20, 40, 60, 120, 180, 240, 300, 360, 420, and 480 min and cells were washed with 1 × PBS (Life Technologies, Darmstadt, Germany) before protein and mRNA lysates were isolated. The MTT viability assay was performed as previously described (Malz et al., 2014 (link)).