Dendritic Cell Differentiation and Activation

Bone marrow cells were obtained from C57BL/6 mice and were separated and prepared as single-cell suspensions; erythrocytes were lysed with ACK buffer. The residual bone marrow cells were cultured in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (HyClone), 100 IU/ml penicillin (Gibco), 1% sodium pyruvate (Corning), and 1% HEPES in the presence of 50 ng/ml rm-GM-CSF (Peprotech) and 2.5 ng/ml rm-IL-4 (Peprotech) to induce the differentiation of dendritic cells. After 6 days of incubation at 37 °C and 5% CO₂, the dendritic cells were identified by using FACS staining with specific fluorescence-labeled CD11c antibody (Biolegend). The dendritic cells were incubated with ALD-DNA overnight with or without additional NaCl (20 mM, Sigma)^{31 (link),32 (link),70 (link),101 (link)} and with or without the STAT1 inhibitor fludarabine (2 μg/ml, Selleck-21679-14-1) and p38 MAPK kinase inhibitor (5 μM, InvivoGen-tlrl-sb20). The harvested dendritic cells were collected for transfer to C57BL/6 mice or for flow cytometric measurement of activation and/or maturation markers.