Af676

Example 9

HEK293/17-IF Cells (MAB Discovery GmbH) were cultivated in DMEM, 10% FCS, 20 μg/ml hygromycin for 5 days before they were seeded out in 384-well white, flat bottom, cell culture treated plates (Corning) at a cell density of 30,000 cells/well in 20 μl medium. Cells were incubated over night at 37° C./5% CO₂. Medium was removed by aspiration and various concentrations of monoclonal or polyclonal (goat-anti-human-IL-1R3, AF676, R&D Systems) antibodies were added in a volume of 10 μl medium. Plates were incubated for 60 minutes at 37° C./5% CO₂. Recombinant human IL-36g (R&D Systems) protein was added in 10 μl medium to a final concentration of 15 ng/ml and plates were incubated for 5 hours at 37° C./5% CO₂. 20 μl Steady-Glo™ (Promega) solution was added to each well, mixed thoroughly and plates were incubated for 10 minutes at room temperature before luminescence was read using a Tecan M1000 plate reader. Fitting curves and EC50 calculation were done using Excel (Microsoft) and XLfit (IDBS).

Example 13

HEK293/17-IF Cells (MAB Discovery GmbH) were cultivated in DMEM, 10% FCS, 20 μg/ml hygromycin for 5 days before they were seeded out in 384-well white, flat bottom, cell culture treated plates (Corning) at a cell density of 30,000 cells/well in 20 μmedium. Cells were incubated over night at 37° C./5% CO₂. Medium was removed by aspiration and various concentrations of monoclonal or polyclonal (goat-anti-human-IL-1R3, AF676, R&D Systems) antibodies were added in a volume of 10 μl medium. Plates were incubated for 60 minutes at 37° C./5% CO₂. Recombinant human IL-36g (R&D Systems) protein was added in 10 μl medium to a final concentration of 15 ng/ml and plates were incubated for 5 hours at 37° C./5% CO₂. 20 μl Steady-Glo™ (Promega) solution was added to each well, mixed thoroughly and plates were incubated for 10 minutes at room temperature before luminescence was read using a Tecan M1000 plate reader. Fitting curves and EC50 calculation were done using Excel (Microsoft) and XLfit (IDBS).