Quantification of Inflammatory Cell Death

BAL was collected two hours after intratracheal instillation of 20 μg LT or vehicle. Cells were stained with fluorescently labeled caspase inhibitors FAM-YVAD-FMK (caspase-1) or FAM-DVED-FMK (caspase-3) and with 7-Amino-Actinomycin (7-AAD, BD Bioscience, Franklin Lakes, NJ USA) according to manufacturer’s instructions (Cell Technology, Inc., Mountain View, CA, USA). For whole lung cell suspension, isolated lungs were cut into pieces and incubated for 1 h in digestion medium (RPMI, collagenase IV 12 mg/ml, DNase I 10mg/ml) at 37°C. A single cell suspension was prepared by passing through 70 μm cell strainer (BD Bioscience, Franklin Lakes, NJ USA). Red blood cells were removed by incubation in lysis buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA). Cell were incubated with Fc block, and stained for CD45, Mac-I, and GR-1 or CCR-2 (all from BD Bioscience, Franklin Lakes, NJ USA). FACS analysis was done using a Beckman Coulter CyAn ADP Analyzer (Beckman Coulter, Inc., Brea, CA). Data were analyzed using FloJo (TreeStar, Ashland, OR) software.