Avanti mini extruder apparatus

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).

Liposomes were formed by lipid (DMPC and DPPC) solubilization with a mixture of chloroform and methanol (1:1, v/v), dried under a stream of nitrogen in a conical tube, and kept under high vacuum for 12 h to remove traces of organic solvent. The suspension (10 mg/mL) was subjected to three freeze/thaw cycles to promote the formation of larger lipid aggregates. The lipid aggregates were then extruded at 40 °C through a polycarbonate membrane, with an etched pore size of 100 nm and 200 nm, using the Avanti Mini-Extruder apparatus (Avanti Polar Lipids) The final concentration of liposomes in the different formulations was 1 mg/mL. The size distribution of the liposomes was determined by a dynamic light scattering analysis (DLS) using a Zetasizer NanoZS (Malvern Instruments, Malvern, UK) in triplicate at 25 °C for 60 s, with an average count rate of 280 kcps [76 (link),77 (link)].