Hi,
I usually differentiate my BMDM with RPMI complete + M-CSF and it works perfect. After a few days you can see the macrophages attached to the plate and after approx. 6-7 days the confluency is high enough to polarize them and to harvest them.
Now I also tried it with RPMI complete + GM-CSF (25ng/ml). But after 4 days nearly no cells are differentiated to macrophages and attached to the plate.
I plated 0.9 x 106, 0.7 x106 and 0.5 x106 cells into different 6-well plates containing 2 ml medium per well.
Why are they not growing/differentiating with the GM-CSF?
Does someone have experience with GM-CSF derived macrophages?
If someone could send me a protocol it would be awesome.
Thank you!
Hi,
I differenciated to macrophages after 6-7 days using GM-CSF and discard and wash to remove cells there are on suspension (dendritic cells).
GM-CSF works good for human and mouse macrophages. They are round and like a fried egg. Functionally similar to alveolar macrophages. Check your GM-CSF. We add GM-CSF 10-20-30 ng/ml.
To my knowledge, human monocytes are differentiated to macrophages (M1) using GM-CSF. For the generation of dendritic cells (immature) a combination of GM-CSF and IL-4 is needed in the human system. However, if you use GM-CSF in the murine system, you generate dendritic cells, already by stimulating monocytes with GM-CSF only.
Hi thank you all for your answers!
Now they are growing nice. They were just much slower than the cell I incubated with M-CSF. I was able to harvest the cells with M-CSF after 6/7 days.. The cells with GM-CSF were ready for harvesting after 12/13 days under the same conditions. And I got a mix of dendritic cells and macrophages with GM-CSF.
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