Anti cd11c immunomagnetic bead purification

DC were generated from freshly-isolated B6 bone marrow (BM) cells, as described.^{22 (link)} On day 2 of culture, either RAPA (LC Laboratories, Woburn, MA) or AZD2014 (Selleck Chemicals, Houston, TX), prediluted in RPMI-1640, was added at the concentration indicated and refreshed on days 4 and 6. Control cultures with no drug administration were set up in parallel. On day 7, cells were collected, counted and CD11c⁺ cells isolated by anti-CD11c immunomagnetic bead purification (Miltenyi Biotec, Auburn, CA). In some experiments, the DC were stimulated for 16–18 h with lipopolysaccharide (LPS) (Salmonella minnesota, 10 ng/ml; R595, Alexis Biochemicals, San Diego, CA).

DC were generated from freshly-isolated femoral BM cells as described.^{47 (link)} Ten million BM cells were cultured in RPMI-1640 with recombinant mouse granulocyte-macrophage colony-stimulating factor and mouse IL-4 (both 1000 U/ml; R & D Systems, Minneapolis, MN). Fresh GM-CSF and IL-4 were added every 2 days. On day 7, the cells were collected, counted and CD11c⁺ cells isolated by anti-CD11c immuno-magnetic bead purification (Miltenyi Biotec, Auburn, CA).