Murine Dendritic Cell Generation Protocol

Cells were generated from naïve mice as previously described (28 (link)). Briefly, bone marrow was removed from femurs and tibiae. After red blood cell lysis (eBioScience, San Diego, CA, USA), the total bone marrow cells (2.5 × 10⁵ cells/ml) were cultured in RPMI 1640 supplemented with 5% heat-inactivated FBS, 10 mM HEPES, 20 µg/ml gentamycin, 100 U/ml penicillin–streptomycin, 2 mM l glutamine and 50 µM 2-ME (Gibco, Invitrogen, Burlington, ON, Canada). Complete medium was complemented with 20% GM-CSF from a mouse GM-CSF-transfected cell line (Ag8653) as a source of GM-CSF (35 (link)). Cells were cultured for 7 days at 37°C in a 5% CO₂ incubator, and fresh medium was added on days 3 and 5. On day 7, clusters were harvested and subcultured overnight to remove adherent cells. Non-adherent cells were harvested on day 8, washed, and used as immature DCs for the studies in complete medium containing 5% GM-CSF. Cell purity routinely comprised 86–90% CD11c^high, as determined by FACS analysis and as previously reported (28 (link)).