Bio plex magpix platform

At 6 or 24 h after instillation of S. pneumoniae (5.8-15 × 10⁶ CFUs/mouse) in Nrf2 null and WT mice, the pulmonary vasculature was flushed with PBS, and a single BAL was performed by instilling 0.9 ml PBS containing 2 mM EDTA per 22 g mouse body weight into the lungs through the trachea. BAL fluid was spun at 300 x g at 4°C for 5 minutes, and the cell-free supernatant was collected and stored at -80°C until use. Thawed samples were spun at 15,000 x g for 2 minutes at 4°C, and the supernatant was collected and aliquoted for use in subsequent assays. Total protein was measured using the Bicinchoninic Acid Protein Assay according to the manufacturer’s instructions (Sigma-Aldrich or Thermo Fisher Scientific, Waltham, MA). The levels of 23 cytokines/chemokines were measured using a multiplex assay on the Bio-Plex MAGPIX platform (Bio-Rad, Hercules, CA, USA). The levels of soluble RAGE, S100A8 and S100A9 were measured using a custom multiplex magnetic bead-based assay (R&D Systems, Minneapolis, MN) on the Luminex platform (Luminex, Austin, TX). Soluble ICAM-1 (sICAM-1, CD54) was measured using an ELISA (R&D Systems). Extracellular double-stranded DNA was measured using Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies, Grand Island, NY).