Cell Culture of Bone Marrow-Derived Dendritic Cells (BMDCs)

BMDCs were generated as described by Lutz et al. (48). Briefly, 2 × 10⁶ bone marrow cells from C57BL/6, C3H/HeN, or C3H/HeJ mice were seeded in 10-mm bacteriological culture dishes in RPMI (HyClone, Logan, Utah, USA) medium supplemented with 20 ng/mL mouse recombinant GM-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured for 10 days, after which they were collected via gently pipetting in preparation for incubation with hemocyanins. For most experiments, BMDCs were seeded at 1 × 10⁶ cells/mL in 6-well plates and incubated with the hemocyanins (1 mg/mL each) for 24 h. Then, the supernatants were collected to measure the cytokine levels, whereas the cells were treated with PBS supplemented with 10 mM EDTA and 4 mg/mL lidocaine and washed twice with FACS buffer in preparation for the maturation marker analysis by FACS. LPS from Escherichia coli Serotype R515 (Enzo Life Sciences) and LPS from Salmonella enterica serotype typhimurium (Sigma-Aldrich) and zymosan A (Sigma-Aldrich), were used as positive controls; PBS or unstimulated cells were used as a negative controls. The TLR2 control Pam3CysK4 was from Invitrogen Life Technologies (Waltham, MA, USA). The TLR9 control CpG oligonucleotide was from Oligos (Wilsonville, OR, USA).