Generation of GM-CSF and FLT3L Dendritic Cells

BMDCs were prepared from 6–10 week-old C57BL/6J female femurs and tibias as previously described.²⁴ (link) Briefly, bone ends were cut off and bone marrow was flushed with RPMI medium (GIBCO, 21875–034) containing 5% FCS and 50 mM of 2-mercaptoethanol (GIBCO, 31350–010). Red blood cells were removed by 1 min exposure to 1xRBC lysis buffer solution (eBioscience, 00–4333–57). For GM-DCs, 3 × 10⁶ cells were seeded onto 6-well plates in 5 mL medium containing 0.8 % supernatant of the J558L GM-CSF producing cell line. Medium was changed at day 2.5 and GM-DCs were ready to use at day 5. For FL-DCs, 8 × 10⁶ cells were seeded onto 6-well plates in 4 mL medium containing 3% supernatant of the B16 FLT3 Ligand producing cell line. FL-DCs were ready to use at day 8.5–9 without any change of medium.