S aureus peptidoglycan pgn

Lipid vesicles were formed using bacterial lipids extracted as described above or commercially available synthetic lipids. The lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1.2-dioleyl-sn-glycero-3-phosphoglycerol (DOPG), and 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (CL), were obtained from Avanti Polar Lipids, Inc (Alabaster, AL) and used without further purification. Desired mixtures of phospholipids were first dried in glass tubes under nitrogen and then maintained under a reduced pressure for at least 60 min. Lipid films were subsequently hydrated in PBS to yield a lipid concentration of 10 mg.mL^-1 for tryptophan fluorescence experiments, 1 mg.mL^-1 for DSC experiments, and in PB at 1 mM for CD experiments. The resulting dispersions of large multilamellar vesicles (MLVs) were subjected to 3 to 5 freeze-thaw cycles and then extruded 15 times through polycarbonate membranes (100 nm pore size, Whatman, Maidstone, UK) on an Avanti mini-extruder apparatus (Avanti Polar Lipids, Inc., Alabaster, AL) to generate large unilamellar vesicles (LUVs). Lipid vesicles were used either alone, or in combination with S. aureus peptidoglycan (PGN) (Sigma).

Neutrophils were purified from whole blood by negative magnetic selection (Miltenyi Biotech). Purified neutrophils were suspended at 1 × 10⁶ cells/mL in Sytox Green solution (Thermofischer, 2.5 μM) and preincubated for 30 min with either 5 ng/ml TNF-α, 10 ng/ml LPS, 2.5 µg/ml CL097 (Invivogen), or medium. These concentrations are those classically used to prime various neutrophil functions, and they are not sufficient to trigger NET release alone. Then, cells stimulated with either 25 nM phorbol myristate acetate (PMA, Sigma), 1 µM fMLF, or 5 μg/ml S.aureus peptidoglycan (PGN, Sigma-Aldrich) for 3 h at 37 °C. DNA release was quantified over time by fluorimetry.