Bone Marrow-Derived Dendritic Cells Generation: A Method to Generate Dendritic Cells from Mouse Bone Marrow

1. Generate bone marrow-derived dendritic cells (BM-DCs).

1. Isolate bone marrow from the femurs and tibias of WT or factor B (fB)^-/- mice on the B6 background. Briefly, collect the tibia and femur from both legs using forceps and scissors. Put them in ice-cold RPMI containing 1% FCS and 100 U/mL penicillin/streptomycin and 2 mM L-glutamine (1% RPMI) in 50 mL conical tubes. Clean the femur and the tibia bones thoroughly by removing all the muscle tissues using forceps and scissors. Transfer the femur and the tibia from the 50 mL conical tubes to a 92 mm diameter Petri dish containing 1% RPMI. Using scissors, cut the ends of the femur or tibia. Fill a 50 mL tube with 25 mL of 1% RPMI 1640 medium. Use this to fill a 3 mL syringe attached to a 26 G needle with 3 mL of 1% RPMI. Insert this syringe into the bone and push the plunger to flush the bone marrow (BM) out of the cavity into the collection tube with 1% RPMI (~3 mL/bone). Spin the cells at 800 x g for 5 min.
2. Resuspend the pellet in 5 mL of ACK lysing buffer for red blood cell lysis. Incubate the cell suspension for 5 min on ice. Add 10 mL of 1% RPMI to the cell suspension to stop the lysis and centrifuge at 800 x g for 5 min at 4 °C. Discard the supernatant.
3. Resuspend the cell pellets in 10 mL of culture media (RPMI 1640 containing 10% FBS, 100 U/mL of penicillin/streptomycin, 2 mM l-glutamine, and 50 mM β-mercaptoethanol) and adjust the volume to meet the final concentration of 2 x 10⁶ cells/mL.
4. Add 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF) to the cell suspension. Culture the bone marrow cells in 100 x 15 mm Petri dishes at 37 °C in 5% CO₂ for 6 days.
5. Replace half of the ongoing culture media (about 5 mL) with fresh media containing 40 ng/mL GM-CSF on day 3.
6. Prewarm the culture media in a water bath at 37 °C. Collect about 5 mL of the media from the bone marrow culture dishes. Centrifuge at 800 x g for 5 min at 4 °C. Discard the supernatant. Resuspend the cell pellet in 5 mL culture media containing 40 ng/mL GM-CSF.
7. Add 25 µg/mL lipopolysaccharide (LPS) to the media on day 6 of the culture to mature the BM-DCs. Save an aliquot of 2 x 10⁶ cells to determine the DC differentiation efficacy by flow cytometry.
8. Collect matured BM-DCs: Use cell lifters to softly scrape DCs from the Petri dishes. Collect all the cell suspensions in 50 mL conical tubes. Centrifuge at 800 x g, for 5 min at 4 °C. Discard the supernatant. Wash the cell pellets 3x with 50 mL of ice-cold PBS. Save about 2 x 10⁶ BM-DCs for immunological phenotype analysis by flow cytometry.