Anti il 1α neutralizing antibodies

ELISA was used for quantification of cytokines in cell-free supernatant and lavage fluid. Cells were centrifuged at 300× g for 5 minutes and the supernatant was collected and stored at −80°C. Murine IL-1α and IL-1β ELISA kits were purchased from R&D Systems. A Biotek Cytation-5 plate reader was used to quantify concentrations.
HEK-blue IL-1R1 cell-line used to measure IL-1α and IL-1β bioactivity was purchased from InvivoGen. For each experiment, 2.8×10⁵ HEK IL-1R1 reporter cells/mL in DMEM (GIBCO) complete media were seeded in 180 μL per well. Twenty μL of each sample was added in duplicates without neutralizing antibodies, with anti-IL-1α neutralizing antibodies (Fisher Scientific), anti-IL-1β neutralizing antibodies (R&D Systems), or both and incubated overnight at 37°C with 5% CO₂. The supernatant was collected and incubated with QUANTI-Blue (InvivoGen) for 30 minutes. SEAP detection and concentration calculations were measured on the Biotek Cytation-5 instrument. IL-1α and IL-1β concentrations were calculated based on a set of standards of known bioactive IL-1α and IL-1β concentrations and presented as pg/mL.