Bone Marrow Cell Differentiation

BM cell differentiation was done according to [24 (link)] with little modifications. Briefly, single cell suspensions were generated from wildtype and Bcl3^-/- BM cells. The cells were suspended in DMEM medium supplemented with 10% FCS, 1% L-glutamine, 1% sodium pyruvate, 1% MEM-NEAA and 1% penicillin/streptomycin, 55 mM 2-mercaptoethanol and Flt3L (100 ng/ml) (Cat no. RP-8665, Invitrogen, Thermo Fischer Scientific) then were cultured at 37°C in a humidified atmosphere at 5% CO2. On day 3, the cells were transferred to a single well containing a monolayer of mitomycin C (Cat no. 50-07-7, Millipore Sigma, Merck, Darmstadt, Germany)-treated OP9 or OP9-DL1 cells, or else were kept unaltered. At Day 7, the cells were supplemented with murine rIFN-γ (40 μg/ml) (Recombinant Murine IFN-γ, Catalog Number:315–05, Peprotech, East Windsor, NJ, USA) or were kept unaltered. At Day 9, all the cells were harvested and used for DC phenotyping by flow cytometry or were used further in the antigen presentation assay described above.