I isolated and differentiated bone marrow-derived macrophages from mice. After differentiation I frozen them and kept them in liquid nitrogen. Then, I thaw them and let them recover for 3-5 days before polarization.
They proliferate perfectly with the differentiation medium (DMEM + 20% FBS + 30% L929 SN), and I can keep them in culture for, at least, 2 weeks after thawing. To induce polarization I split the cells, count and seed them at a density of 100-150,000 cells/cm2 in classical medium (DMEM + 10% FBS). I culture them for 24h with this medium, after I change the medium to the one with the specific cytokines to induce anti- and pro-inflammatory polarization (IL-10 and protein homogenate from injured muscle, respectively) for 48h. After 72h from seeding, I get a viability of 10-15%, which is too low.
I have several questions for which I haven't found a clear answer yet.
- Can the incubation of 24h with classical medium before polarization can affect BMDM viability? Should I reduce this time?
- Is it possible to induce anti- and pro-inflammatory polarization with the presence of M-CSF (L929 SN)? I have read that the M-CSF promotes and anti-inflammatory phenotype. Would it be possible to induce a pro-inflammatory state even with the presence of M-CSF)
I attach the photos from the BMDM after 24h of seeding with differentiation or classical medium.
Hope you can help me!
Thanks :)
1-It does not affect the polarization property of macrophages. The incubation of 24 hours with the classical medium before polarization does not negatively impact BMDM viability or alter their ability to undergo polarization. This incubation time is commonly used to allow macrophages to adhere and recover from any stresses induced during isolation. As a result, it does not interfere with the subsequent polarization process and allows the cells to maintain their normal function during polarization.
2-M-CSF (Macrophage Colony-Stimulating Factor) plays a critical role in promoting the differentiation of monocytes into macrophages and is involved in developing macrophages with an anti-inflammatory or M2 phenotype. However, it is essential to note that M-CSF alone typically results in a basal level of anti-inflammatory properties in macrophages, and it does not induce a robust pro-inflammatory response.
To induce a pro-inflammatory state in macrophages, it is common to use other stimuli, such as pro-inflammatory cytokines like interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). These stimuli promote the polarization of macrophages into the M1 or pro-inflammatory phenotype, characterized by the secretion of pro-inflammatory cytokines like TNF-alpha, IL-1beta, and IL-6. Add other factors like IL-4 and IL-13 along with M-CSF to induce a pro-resolving or anti-inflammatory phenotype.
Hi All! I want to ask whether should I replenish the media during 5-6 days of incubation w M-CSF?
Hi Fulya Koksalar Alkan ! I don't do it and my cells differentiate perfectly into BMDM, but I read that some other people add more medium at day 3. Not sure which protocol is the best
is DMEM is right media for polarisation of Bone marrow derived Macrophage
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