Bone Marrow Derived Dendritic Cell (BMDC)

Bone marrow cells were harvested from tibiae and femurs of mice, erythrocytes were lysed with ACK lysis buffer, filtered and cultured in VLE-DMEM media (FG1445; Merck Millipore) supplemented with 5% culture supernatants containing murine GM-CSF (prepared in house from X63-GM-CSF cell line culture), or 20% of supernatant containing murine FLT3L (prepared in house from CHO FLT3-L FLAG cell culture; CHO FLT3-L FLAG cell line was kindly provided by Tim Sparwasser Institute of Infection Immunology, TWINCORE, Hannover, Germany), heat inactivated 10% fetal calf serum (10270; Gibco), 1% of penicillin, streptomycin, L-glutamine solution (G6784 Sigma), and 0.1% 50mM beta mercaptoethanol (D4551, Sigma), which constitute a complete media (CM) and cells were incubated at 37°C in a humidified atmosphere with 5% CO₂. Bone marrow cells seeded with 3 × 10⁶ cells per petri dish (58 cm²) in 10 ml of CM or in 24-well plates with 2 × 10⁵ cells/well in 1 ml CM. On day 3, extra CM was added with volume equal to initial volume. On days 5–6, cells were either harvested for seeding in 24 well plates or directly treated in petri dishes.