Protocol for generating mouse dendritic cells from bone-marrow progenitor cells used in our Nature paper.
Method Article
Generation of mouse bone marrow-derived dendritic cells (BM-DCs)
https://doi.org/10.1038/nprot.2009.137
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Protocol for generating mouse dendritic cells from bone-marrow progenitor cells used in our Nature paper.
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
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 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.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.
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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