Bone Marrow-Derived Dendritic Cell Assay

The animal studies were reviewed and approved by the University of Kansas Institutional Animal Care and Use Committee practices (protocol AUS 221-03). Bone marrow dendritic cells (BMDC) were isolated as previously described (34). Briefly, BALB/c mice (Charles River Laboratories, Wilmington, MA) were euthanized and femurs were extracted. The ends of the femurs were cut off and the bone marrow flushed with cold media. After harvesting the cells by centrifugation, they were incubated with RBC lysis buffer (Sigma), neutralized with complete RPMI and filtered with a 70 µm filter. The resulting single cell suspension was grown in presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (Invitrogen) at a concentration of 20 ng/ml of complete RPMI. On days 3 and 6, 50% of the media was replaced with fresh complete RPMI containing 20 ng/ml of GM-CSF. On day 8, loosely adherent cells (BMDCs) were harvested. The harvested cells were seeded into 6 well plates (10⁶ cell/well) and treated with recombinant proteins at a low, medium and high antigen dose. After 32 h, cells were harvested, blocked and stained with fluorochrome tagged antibodies. The cells were then analyzed by flow cytometry.