Recombinant human il 1α or il 1β

Example 11

A549 cells were seeded out at a density of 6,000 cells/well in 25 μl medium in 384-well clear cell culture treated plates (Corning) in DMEM, 10% FCS, 1% Pen/Strep. Cells were incubated over night at 37° C./5% CO₂. Medium was removed by aspiration and monoclonal or polyclonal (goat-anti-human-IL-1R3, AF676, R&D Systems) antibodies were added at various concentrations in a volume of 12.4 μl medium and incubated for three hours at 37° C./5% CO₂. Recombinant human IL-1α or IL-1β (R&D Systems) proteins were added in 12.5 μl medium to a final concentration of 0.1 ng/ml and plates were incubated for 48 hours at 37° C./5% CO₂. Secreted human-IL-6 levels in the cell supernatant were measured using the DuoSet human IL-6 ELISA kit (R&D Systems, Cat. No. DY206-05) according to the manufacturer's instructions. Fitting curves and EC50 calculation were done using Excel (Microsoft) and XLfit (IDBS).

Example 10

A-549-NFκB-RE-Luc (Signosis) were cultivated in DMEM, 10% FCS, 1% Pen/Strep for 5 days before they were seeded out in 384-well white flat bottom polystyrene tissue-culture-treated microplates (Corning) at a cell density of 40,000 cells/well in 25 μl medium. Cells were incubated over night at 37° C./5% CO₂. Medium was removed by aspiration and monoclonal or polyclonal (goat-anti-human-IL-1R3, AF676, R&D Systems) antibodies added at various concentrations in a volume of 10 μl medium and incubated for 60 minutes at 37° C./5% CO₂. Recombinant human IL-1α or IL-1β (R&D Systems) proteins were added in 10 μl medium to a final concentration of 0.1 ng/ml and plates were incubated for 5 hours at 37° C./5% CO₂. 20 μl Steady-Glo™ (Promega) solution were added to each well, mixed thoroughly and plates were incubated for 10 minutes at room temperature before luminescence was measured using a Tecan M1000 plate reader. Fitting curves and EC50 calculation were done using Excel (Microsoft) and XLfit (IDBS).

Example 7

A549 cells were seeded out at a density of 6,000 cells/well in 25 μl medium in 384-well clear cell culture treated plates (Corning) in DMEM, 10% FCS, 1% Pen/Strep. Cells were incubated over night at 37° C./5% CO₂. Medium was removed by aspiration and monoclonal or polyclonal (goat-anti-human-IL-1R3, AF676, R&D Systems) antibodies were added at various concentrations in a volume of 12.5 μl medium and incubated for three hours at 37° C./5% CO₂. Recombinant human IL-1α or IL-1β (R&D Systems) proteins were added in 12.5 μl medium to a final concentration of 0.1 ng/ml and plates were incubated for 48 hours at 37° C./5% CO₂. Secreted human-IL-6 levels in the cell supernatant were measured using the DuoSet human IL-6 ELISA kit (R&D Systems, Cat. No. DY206-05) according to the manufacturer's instructions. Fitting curves and EC50 calculation were done using Excel (Microsoft) and XLfit (IDBS).