Mouse immunosuppressive dendritic cells (University of Pittsburgh)

Bone marrow is obtained from the femurs and tibiae of female NOD mice. The red blood cells are lysed using a commercially available reagent and the bone marrow cells are plated in 24-well multiwell plates at 2 × 10⁶ cells/ml in R-10 medium (RPMI 1640, heat-inactivated FBS (10%)  2-ME (50 μM), sodium pyruvate (1%), nonessential amino acids (1%), penicillin-streptomycin solution (1%) with the addition of GM-CSF (4 ng/ml) and IL-4 (1000 U/ml). Two days later, the nonadherent cells are removed and a 1:1 volume of conditioned medium, fresh R-10 medium, and cytokines are added to the adherent cells. Three days later, the loosely adherent cells are gently agitated and harvested. The antisense oligonucleotides (AS-ODN) mixture consisted of phosphorothioate-modified ODNs each targeting the 5′ end of the CD40, CD80, and CD86 primary transcripts. The sequences were: CD40 AS-ODN, 5′-CAC AGC CGA GGC AAA GAC ACC ATG CAG GGC A-3′; CD80 AS-ODN, 5′-GGG AAA GCC AGG AAT CTA GAG CCA ATG GA-3′; CD86 AS-ODN, 5′-TGG GTG CTT CCG TAA GTT CTG GAA CAC GTC-3′. Cells are treated 18–24 h in 10% heat-inactivated FBS/RPMI 1640 with a mixture of  CD40 AS-ODN (3.3 μM), CD80 AS-ODN (3.3 μM), and CD86 AS-ODN (3.3 μM). The cells are then washed extensively in PBS.