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Cell Culture and Transfection
THP1 monocytes (American Type Culture Collection) and human aortic endothelial cells (HAECs) obtained from Cell Applications Inc. (San Diego, CA) were cultured and maintained as described.5 Complete sequence of human UCP2 was amplified, inserted into the expression vector pcDNA3.1(Invitrogen), and confirmed by DNA sequencing. L-{alpha}-phosphatidylcholine (ePC), L-{alpha}-phosphatidylserine (bPS), 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine (DOPE), and sphingomyelin (eSph) and cholesterol (Chol) (Avanti Polar Lipids, Alabaster, AL) in chloroform, were mixed in an approximate weight ratio of ePC:DOPE:eSph:Chol:bPS=1.6:3:1.47:3:1.3 (10.37 mg/mL), filtered (0.2 µm pore size), dried in a N2-filled rotary evaporator to make a thin film, and mixed with 200 µg UCP2-pcDNA3.1 (1 µg/µL stock). Hemagglutinating virus of Japan (HVJ) was prepared as described.9 Briefly, HVJ was irradiated by ultraviolet light (198mJ/cm2) to inactivate the RNA genome, and 1 mL HVJ suspension (20,000 hemagglutinating units) was mixed with the liposome-UCP2-pcDNA3.1 complex. The resultant liposome-UCP2-pcDNA3.1-HVJ complex was separated from free HVJ by centrifugation on a discontinuous 30% sucrose gradient (25,000 rpm at 4°C for 2 hours), and then collected and suspended in 2 mL balanced salt solution. Liposome-UCP2-pcDNA3.1-HVJ complex (250 µL) was added dropwise to 5 million THP1 monocytes in 20 mL RPMI medium 1640 containing 2% heat-inactivated FBS. After 24 hours, monocytes were harvested and used for further analysis. Flow cytometry analysis of monocytes stained with propidium iodide showed that the liposome complex was not cytotoxic.

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