40pg/uL human IL-1RA (PeproTech, US) was pre-incubated with 5ng/uL antibody (G4–21 or isotype control. IL-1RA and antibody were applied to HEK-Blue IL-1β cells (InvivoGen, US) for 2 hours and stimulated with 2pg/uL human IL-1β (PeproTech, US) for 36 hours at 37°C. Alternatively, cells were treated with 8–10 nM IL-1RA and commercial monoclonal (mAbs) or polyclonal antibodies (pAbs) at 40–2000nM, or with patient plasma at 1:10 and 1:20 dilutions, and subsequently stimulated with 0.1–1 nM IL-1β for 24 hours. Cells were treated with antibodies in the presence of IL-1α and IL-1RA, IL-1RA alone, or media, and supernatants assayed using the QUANTI-Blue assay (InvivoGen, hkb-il1b, US).
Human A549 lung epithelial cells and MRC-5 lung fibroblasts (ATCC, US) were treated with 10nM IL-1RA and patient plasma and stimulated with 0.5nM human IL-1α (PeproTech, US) for 24 hours at 37°C. RNA was isolated, cDNA prepared, and samples analyzed using TaqMan FAM-conjugated primer sets. Supernatants were collected and assayed for human IL-6 (R&D, US), IL-8 (R&D, US), and G-CSF (R&D, US) via ELISA.
Human A549 lung epithelial cells and MRC-5 lung fibroblasts (ATCC, US) were treated with 10nM IL-1RA and patient plasma and stimulated with 0.5nM human IL-1α (PeproTech, US) for 24 hours at 37°C. RNA was isolated, cDNA prepared, and samples analyzed using TaqMan FAM-conjugated primer sets. Supernatants were collected and assayed for human IL-6 (R&D, US), IL-8 (R&D, US), and G-CSF (R&D, US) via ELISA.