Protocol for generating mouse macrophages from bone-marrow progenitor cells used in our Nature paper.
Method Article
Generation of mouse bone marrow-derived macrophages (BM-MFs)
https://doi.org/10.1038/nprot.2009.136
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Protocol for generating mouse macrophages from bone-marrow progenitor cells used in our Nature paper.
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
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 x 106 cells in 100 x 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 x 106 cells in 100 x 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.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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