Bone Marrow-Derived Dendritic Cell Culture

Bone marrow stem cell progenitors were obtained from the femurs and tibiae of naïve BALB/c mice (n = 3) and cultured in complete medium (CM) consisting of RPMI (1640 with L-glutamine, Lonza, Basel, Switzerland) supplemented with 10% FBS; a mixture of antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin, Lonza); and 10 mM HEPES (Lonza) in the presence of 20 ng/mL murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, London, UK), as previously described [54 (link)]. Fresh medium containing GM-CSF was added to the cultures every 3 days. On day 7, half of the volume was removed, and cells were centrifuged at 500× g for 10 min. The pellet was then resuspended on CM with freshly added growth factor and cultured at 37 °C with 5% CO₂. On day 10, nonadherent cells were collected and considered BMDCs based on the expression of CD11c as described before [20 (link)].