J774.2 macrophage culture

J774.2 murine macrophages were cultured in T25 tissue culture flasks containing 5 ml of DMEM buffered with sodium bicarbonate and supplemented with 10% FBS. Cells were grown to 80% confluency at 37 °C in a 5% CO₂ incubator and passaged by cell scraping. Cells were cultured for no more than six generations, at which time new cells were revived from liquid nitrogen stocks. For microscopy experiments, no. 1.5 thickness, 18 mm diameter round coverslips were placed into the wells of a 12-well tissue culture plate, 1 ml of DMEM+10% FBS added to each wells and 0.2 ml (~2.5 × 10⁵ cells per well) of the above cell suspension added to each well. Cells were cultured for at least 8 h before transfection. Transfection with DNA vectors or siRNA was conducted using FugeneHD, as per the manufacturer's instructions. Briefly, for each well in a 12-well plate, 1.1 μg of DNA or 0.75 μg of siRNA was diluted into 100 μl of serum-free DMEM, followed by 3.3 μl of FugeneHD. The resulting mixture was incubated for 20 min at room temperature, and then added dropwise to the J774.2 cells. Cells were incubated for at least 18 h at 37 °C in a 5% CO₂ before imaging.