The N9 and primary microglia activation was achieved by exposure to LPS at 0·1, 0·5 or 1 μg/ml, for different periods of time, ranging from 30 min to 18 hr. The delivery liposomal system (DLS) cationic liposomes were prepared by mixing 1 mg DOGS with 1 mg DOPE in 40 μl 90% ethanol, followed by the addition of 360 μl H2O, as described previously.21 After homogenization, the mixture was incubated for at least 30 min to allow liposome formation. The final lipid Liproxstatin-1 mouse concentration was 5 mg/ml (2·5 mg DOGS and 2·5 mg DOPE). The DLS lipoplexes were prepared by gently mixing 10 μg anti-miRNA
oligonucleotides with 190 μg total lipid in HEPES-buffered saline solution (HBS: 20 mm HEPES, 100 mm NaCl, pH 7·4) at a final volume of 1300 μl, followed by incubation for 30 min at room temperature. Cationic liposomes composed of DOTAP : DOPE (1 : 1 molar ratio) were prepared as previously described
by Campbell.22 Briefly, a mixture of 1 ml DOTAP and 1 ml DOPE in chloroform (from stock solutions of 25 mg/ml DOTAP and 26·6 mg/ml DOPE) was dried under nitrogen to obtain a thin lipid film. The film was dissolved in 100 μl ultrapure ethanol and the resulting ethanol solution was injected with a Hamilton syringe into 900 μl pre-heated (40°) HBS buffer, maintained continuously under vortex. The resulting multi-lamellar vesicles were briefly sonicated to obtain small PLX-4720 concentration uni-lamellar vesicles and diluted with HBS to a final DOTAP concentration of 1 mg/ml. Folate-associated lipoplexes (FA-lipoplexes) were prepared by incubating 41·9 μg DOTAP with 320 μg folate (32 μg/μg pDNA) for 15 min, followed Oxaprozin by addition of 10 μg pDNA at a
final volume of 1 ml in HBS. The mixture was further incubated for 30 min at room temperature. Both liposome formulations were stored at 4° until use and the lipoplexes were used immediately after preparation. Inhibition or over-expression of miR-155 was achieved by delivery of anti-miR-155 oligonucleotides or plasmid DNA encoding miR-155, respectively, to N9 cells. Immediately before transfection, cells were washed and the medium was replaced with Optimem (900 μl/well), free of serum and antibiotics. For inhibition of miR-155, 100 μl DLS lipoplexes containing 14·6 μg lipid and 0·1 nmol (0·772 μg) anti-miR-155 oligonucleotides were delivered to N9 cells, to obtain a final oligonucleotide concentration of 100 nm/well. Parallel experiments were performed using a negative control oligonucleotide sequence to ensure that the modulation of miR-155 targets could be attributed only to the specific anti-miR-155 oligonucleotide and not to the transfection process per se. Delivery of plasmid DNA to N9 cells was achieved through the use of FA-lipoplexes. One hundred microlitres of FA-lipoplexes, containing pmiR-155 were delivered to N9 cells to obtain a final plasmid concentration of 1 μg/well.