Generation of mouse bone marrow-derived macrophages (BM-MFs)
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 macrophages from bone-marrow progenitor cells used in our Nature paper.
Materials
Reagents
M-CSF-transduced L929 cell line.
BMMFs 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 - 30%
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 30% of growth supernatant of M-CSF-transduced L929 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.Upon reaching confluence (approximately 7 days) split adhered cells and seed 5 × 106 cells in 100 × 20 mm in non-treated cell culture plates in 10 ml of conditioned medium.
6.BMMFs are ready for experimental use when the percentage of CD11b+ cells is higher than 90% as measured by FACS analysis.
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
Macrophage differentiation, M-CSF