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MNCF preparation. MNCF, isolated from the supernatant of LPS-stimulated murine macrophage P388-D1 cell line, as described [7], was further purified by affinity chromatography on detoxi-gel beads, to eliminate virtual LPS contamination. The LPS-depleted MNCF is named dtxMNCF only when it BAM7 is compared with crude preparations before detoxi-gel.
Neutrophil isolation and functional analysis. Human neutrophils were prepared as described [9]. For apoptosis, neutrophils (1 × 106/ml, in RPMI, 10% fetal calf serum) were incubated for 18 h at 37 °C with 5% CO2 and stained with 7AAD and PE-labeled Annexin V, according to manufacturer’s instructions. For activation, neutrophils (2 × 106/ml in HBSS2+/0.1% bovine serum albumin, BSA) were preincubated or not with inhibitors and stimulated with agonists, washed with ice-cold PBS, 1% BSA, 0.1% sodium azide, and labeled at 4 °C with FITC-labeled mAbs. For the oxidative burst, neutrophils were preincubated with the cell-permeant fluorescent probe dichlorodihydrofluorescein diacetate, DCFHDA (5 μM; 10 min at 37 °C) before stimulation, as described [10]. Cells were analyzed by flow cytometry on a pelagic zone Becton Dickinson FACSCalibur (Mountainview, CA).