Pe selection magnetic beads

Peritoneal PMN were recovered from Ifnb^+/+ or Ifnb^−/− mice at 24 h PPI, separated using PE-conjugated Gr1 antibodies with PE selection magnetic beads (StemCell Technologies) and incubated (1*10⁶ cells in 1 ml of culture media) with or without IFN-β (20 ng/ml) and/or Q-VAD (10 µM) for 24 h. Bronchoalveolar lavage fluid cells were resuspended in 100 μl PBS. 2*10⁵ cells were stained with Annexin-V-FITC and PI MEBCYTO Apoptosis Kit (MBL Laboratories). Apoptosis was evaluated by flow cytometry using FACSCanto II (BD Biosciences) and analyzed by FlowJo software (Treestar). Alternatively, PMN lysates were prepared after 6 h of culture and immunoblotted for active (cleaved) caspase-3 using specific antibodies (Cell Signaling Technology).
Apoptosis in human PMN was assessed by flow cytometry with FITC-conjugated annexin-V (BD Biosciences) in combination with propidium iodide (Molecular Probes). For nuclear DNA analysis, neutrophils were suspended in 0.2 ml 0.1% sodium citrate solution containing 50 ug/ml propidium iodide and 0.1% Triton X-100 immediately before assay. Mitochondrial transmembrane potential was monitored following neutrophil staining with the lipophilic fluorochrome chloromethyl-X-rosamine (CMXRos, 20 nM, Millipore-Sigma) and the fluorescence was analyzed in a FACSCalibur flow cytometer.

Peritoneal exudates and bronchoalveolar lavage fluid were collected at the indicated times and the levels of IFN-β in cell-free fluids were determined by VeriKine-HS Mouse Interferon Beta Serum ELISA Kit (Pestka Biomedical Laboratories, Inc). In some experiments, macrophages were recovered 66 h post PPI, separated using PE-conjugated anti-F4/80 Abs (1ul per 10*10⁶ cells in 100ul, Biolegend 123110) with PE selection magnetic beads (StemCell Technologies) and incubated (1 × 10⁶ cells in 1 ml of culture media) with TGF-β (5 ng/ml), poly (I:C) (4 µg/ml) or apoptotic cells (1:5 ratio) for 24 h. Then, IFN-β content in conditioned culture media was determined by ELISA.

Peritoneal macrophages were isolated using PE selection magnetic beads (StemCell Technologies) and incubated (5 × 10⁶ cells in 5 ml of culture media) with LPS (1 µg/ml) or vehicle in RPMI 1640 under a humidified 5% CO₂ atmosphere at 37°C. After 24 h, the supernatants were collected, and their TNFα, IL-6, CCL3, and IL-10 contents were determined by standard ELISA (Biolegend kits for TNFα, IL-6, and IL-10 and R&D Systems kit for CCL3).