Primary cells and DNA stimulation

BMMs and dendritic cells were prepared as follows: bone marrow cells were flushed from the femurs and tibias of C57BL/6 mice and subsequently depleted of red blood cells using ammonium chloride. For BMMs, cells were seeded at a density of 1.5 × 10⁶ cells per well in 24-well plates in DMEM supplemented with 20 ng/ml murine M-CSF. Fresh medium was added every 2 days. On day 5, cells were harvested. For BMDCs, cells were seeded at a density of 1 × 10⁶ cells per well in 24-well plates in RPMI-1640 medium supplemented with 20 ng/ml GM-CSF. Fresh medium was added every 2 days. On day 7, cells were collected for analysis.

DNA was purified from the supernatant of SN-38-treated HCT-116 cells or the fluid from CPT-11-treated mice using phenol/chloroform/isoamyl alcohol. The primary cells were primed with 200 ng/ml LPS from Escherichia coli 0111:B4 (Sigma) for 3 h before transfection with DNA at the indicated concentration for 4 h using Lipofectamine 2000 (Invitrogen).