Generation of mouse bone marrow-derived dendritic cells (BM-DCs)
Lab/Group: Granucci Lab (University of Milano-Bicocca)
Related Journal & Article Information
Journal: Nature
Article Title: CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation
Introduction
Protocol for generating mouse dendritic cells from bone-marrow progenitor cells used in our Nature paper.
Materials
Reagents
GM-CSF-transduced B16 cell line.
BMDCs culture medium recipe (conditioned medium):
HI FBS (EuroClone) - 10%
L-Gln (EuroClone) - 2mM
Penicillin/Streptomycin (EuroClone) - 50 U/ml
Beta-mercaptoethanol (EuroClone) - 50 microM
B16-GMCSF growth supernatant - 10%
IMDM (EuroClone) - to volume
Equipment
Procedure
1.Flush mouse tibiae and femurs with ice-cold PBS through a 70 μm-wide cut-off cell strainer.
2.Centrifuge 5’ at 1400 rpm. Resuspend pelleted cells in conditioned medium (supplemented with 10% of growth supernatant of GM-CSF-transduced B16 cells).
3.Seed 7 × 106 cells in 100 × 20 mm non-treated cell culture plates in 10 ml of conditioned medium.
4.Incubate at 37 °C – 5% CO2.
5.On day 4 and 7 add 5 ml of pre-warmed conditioned medium.
6.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.
7.Harvest the supernatant and gently wash the plate once with 5 ml of pre-warmed PBS.
8.Incubate 2’ with 5 ml of 2mM EDTA at 37 °C – 5% CO2.
9.Collect cells, wash once with PBS.
10.Centrifuge 5’ at 1200 rpm and resuspend pelleted cells in conditioned medium.
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
Dendritic cell differentiation, GM-CSF