Anti il 1β 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.

Neutrophils (2x10⁵ /well) or macrophages (3.5x10⁴ /well) were stimulated as indicated. Cell-free supernatants were collected by centrifugation in V-bottom 96-well plates (PlateOne). Proteins were measured by ELISA following manufacturer’s recommendations (DuoSet, R&D).
IL-1β bioactivity was quantified using the HEK-blue IL-1R1 reporter cell-line (InvivoGen). 2.8 x10⁵ cells/mL in DMEM (GIBCO) complete media with or without anti- IL-1β neutralizing antibodies (R&D Systems) and incubated overnight at 37 C with 5%CO2. Supernatants were incubated with QUANTI-Blue (InvivoGen) for 30 minutes, and cytokines were measured on the Biotek Cytation-5 reader. IL-1 concentrations were calculated as pg/ml based on standards.