Swine Dust Extract Inflammation Protocol

Aqueous organic dust extract (ODE) was prepared from settled dust collected from horizontal surfaces 3 ft. above the floor in swine confinement feeding operations. Extracts were batch prepared and filter (0.22 μm) sterilized as previously described [21 (link), 22 (link)]. Stock ODE was diluted to a concentration of 12.5% (vol/vol) in sterile phosphate buffered saline (PBS, pH 7.4, diluent), a concentration previously shown to elicit optimal lung inflammation in mice [21 (link), 22 (link)]. ODE 12.5% contains approximately 3–4 mg/ml total protein as measured by Nanodrop spectrophotometry (NanoDrop Technologies, Wilmington, DE) and endotoxin levels range from 22.1 to 91.1 EU/ml as measured by the limulus amebocyte lysate assay using manufacturer instructions (Sigma). Specific microbial biomarkers inferred from shotgun metagenomics of DNA pyrosequencing reads of the dust samples have been previously detailed [23 (link)].