A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP

McWhirter, Sarah M.; Barbalat, Roman; Monroe, Kathryn M.; Fontana, Mary F.; Hyodo, Mamoru; Joncker, Nathalie T.; Ishii, Ken J.; Akira, Shizuo; Colonna, Marco; Chen, Zhijian J.; Fitzgerald, Katherine A.; Hayakawa, Yoshihiro; Vance, Russell E.

Cell stimulations (transfections). Cells were transfected using Lipofectamine 2000 (LF2000; Invitrogen) according to the manufacturer's protocol. All nucleic acid stimulants were mixed with LF2000 at a ratio of 1 µl LF2000/1 µg nucleic acid, incubated at room temperature for 20-30 min, and added to cells at a final concentration of 3.3 µg/ml (96-well plates) or 4 µg/ml (6-well plates). For pI:C, 2 mg/ml of the stock solution was heated at 50°C for 10 min and cooled to room temperature before mixing with LF2000. Transfection experiments were done for 6 h, unless otherwise stated in the figures. For phosphodiesterase treatment of c-di-GMP, 0.5 µg/µl c-di-GMP was incubated for 2 h at room temperature in Optimem buffer (Invitrogen), with 15 mM MgCl 2 and 1 U Phosphodiesterase I (GE Healthcare).