Quantification of caspase-1 activity
Bronchial epithelial cells were plated in 6-well plates at 5×105 cells/well overnight. Cells were primed with LPS for 5 hours and stimulated with ATP for 1 hour or stimulated with live PAO1 for 3 hours. PBMCs were stimulated the same day as blood donation. PBMCs were seeded in a 96-well plate at a density of 4.5×105 cells/well (2.5×106 cells/ml), primed with LPS for 5 hours and then stimulated with ATP for 1 hour or Poly(dA:dT) for 3 hours or stimulated with live PAO1 for 3 hours. Caspase-1 activity was measured using FLICA (Immunochemistry Technologies), a cell-permeable fluorescent probe (FAM-YVAD-fmk) that binds active caspase-1. Cells were incubated 1 hour with FLICA at 37°C and stained with PE-Cy7-conjugated anti-CD14 antibodies (eBioscience) to identify monocytes. The gating strategy consisted of including live cells that were CD14 positive which were subsequently analyzed for the frequency of FLICA positive cells.