Materials and Reagents

GM-CSF-transduced B16 cell line
HI FBS (EuroClone, catalog number: EC S0180L )
L-Glutamine (EuroClone, catalog number: EC B3000D )
Penicillin/streptomycin (EuroClone, catalog number: EC B3001D )
IMDM (EuroClone, catalog number: EC B2072L )
Beta-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
B16-GMCSF growth supernatant
Phosphate buffered saline (PBS) (EuroClone, catalog number: ECM9605AX )
EDTA
BMDCs culture medium/conditioned medium (see Recipes)

Equipment

Centrifuges 70 μm-wide cut-off cell strainer
Non-treated cell culture plates
Incubator (37 °C and 5% CO₂)
Fluorescence activated cell sortor (FACS)

Procedure

Flush mouse tibiae and femurs with ice-cold PBS through a 70 μm-wide cut-off cell strainer.
Centrifuge 5 min at 450 x g. Resuspend pelleted cells in conditioned medium (supplemented with 10% of growth supernatant of GM-CSF-transduced B16 cells).
Seed 7 x 10⁶ cells in 100 x 20 mm non-treated cell culture plates in 10 ml of conditioned medium.
Incubate at 37 °C and 5% CO₂.
On day 4 and 7 add 5 ml of pre-warmed conditioned medium.
At day 8/9 the percentage of CD11c⁺ cells should be higher than 90% as measured by FACS analysis. BMDCs are then ready for experimental use.
Harvest the supernatant and gently wash the plate once with 5 ml of pre-warmed PBS.
Incubate 2 min with 5 ml of 2 mM EDTA at 37 °C and 5% CO₂.
Collect cells, wash once with PBS.
Centrifuge 5 min at 250 x g and resuspend pelleted cells in conditioned medium.

Recipes

BMDCs culture medium recipe (conditioned medium)
HI FBS - 10%
L-Gln - 2 mM
Penicillin/streptomycin - 50 U/ml
Beta-mercaptoethanol - 50 μM
B16-GMCSF growth supernatant - 10%
IMDM - to volume

Acknowledgments

This protocol is described in our Nature paper (Zanoni et al., 2009). This work was supported by grants from the CARIPLO Foundation, the European Commission 6th Framework Program (MUGEN and DC-THERA contracts), the European Commission 7th Framework Program (TOLERAGE and ENCITE contracts), the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the and the Italian Ministry of Education and Research (COFIN).

References

Zanoni, I., Ostuni, R., Capuano, G., Collini, M., Caccia, M., Ronchi, A. E., Rocchetti, M., Mingozzi, F., Foti, M., Chirico, G., Costa, B., Zaza, A., Ricciardi-Castagnoli, P. and Granucci, F. (2009). CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation. Nature 460(7252): 264-268.