Recombinant mouse il 1α

Upon arrival, mouse recombinant IL-1α (R&D Systems, Minneapolis, MN, USA) was diluted in sterile phosphate-buffered saline containing 0.1% low endotoxin bovine serum albumin (BSA) (also used as vehicle control). To avoid freeze thaw cycles, the diluted stock solution (50 μg/mL) was then aliquoted and frozen for dilution to the desired dose on the day of surgery or treatment.

For ELISA measurement of intracellular or released pro-IL-1β or IL-1α, fresh lung cells purified from naïve or silica treated mice, primary cultured macrophages, J774, MLg or LA4 (10⁶ cells/well in 96-well plate) were exposed overnight with LPS (0,1μg/ml; Enzo Life Sciences, Antwerpen, Belgium), DQ12, mouse recombinant IL-1α, IL-33, IL-1β, TNF-α (R&D systems, Minneapolis, USA) or HMGB1 (chemokine, non-oxydable and cytokine form; HMGBiotech, Milano, Italia) when necessary. For intracellular cytokine measurement, cell pellets were lysed by addition of 100 μl of triton X-100 0,1%. The ex vivo released of IL-1α were quantified by ELISA in the supernatants of cells purified from silica-treated lungs maintained in culture during 24 hours in 100 μl of DMEM. For in vitro IL-1α release in response to particles, fresh alveolar F4/80⁺ macrophages (10⁶ cells/well in 96-well plate) or J774 cells (0.4 10⁶ cells/well in 96-well plate) were incubated overnight with particles dispersed in 100 μl of DMEM and IL-1α was measured in the supernatant.
For western blot analysis, cells were lysed on ice in 200 μl Triton lysis buffer (TLB) (1% Triton, 25mM Tris pH 7.4, 150mM NaCl + anti-protease tablets from Roche Applied Science at 2 mg/ml). Protocol for Western blot is described elsewhere [84 (link)].