Hi everyone, I'm trying to establish a standard protocol to cultivate BMDMs. I'm culturing BM cells for 7 days in 100mm petri dishes, addition of M-CSF(20ng/mL) on day 0 and day 3.
I would like to confirm if the bone marrow cells on day 7 (with no stimulation added/before start any kind of stimulation) are really differentiated into macrophages. I'll do it through flow cytometry (CD11b, F4/80, CD11c and MHC class II) but I would like also to see at gene expression level. Any recommendations?
Figure 3 of this review paper shows some of the most widely used gene expression markers. Article Gene Regulatory Network Modeling of Macrophage Differentiati...
Dear Antonia Corrêa Castro Ferreira ,
Read the article. It will certainly serve your query.
Link- Article Bone marrow-derived and peritoneal macrophages have differen...
BR, Babu
Dear Antonia,
we recently puplished a small paper, which describes very detailed the cultivation of BMDM and also how to identify the commonly used markers for differentiation (meaning CD11b-F4/80 double positive).
One important suggestion: During cultivation we have M-CSF in the medium from d0 and the medium is not changed. We also add fresh medium with M-CSF on d4.
For the protocol, please have a look, if you like at:
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA, November 2019, Journal of Visualized Experiments
DOI: 10.3791/60143
For examples how our cultivated BMDM react, please have look at:
The β2 Integrin Mac-1 Induces Protective LC3-Associated Phagocytosis of Listeria monocytogenes, March 2018, Cell Host & Microbe 23(3)
DOI:10.1016/j.chom.2018.01.018
and
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO, February 2019, ScienceSignaling12(568):eaar5926
DOI: 10.1126/scisignal.aar5926
Please have also a look in my "Questions section" at d"ifferent macrophage types".
For any further advice or help ask any time.
All the best and stay healthy,
Marc
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