I have come across many studies regarding isolation of monocytes from PBMCs and then culturing these adherent cells. To differentiate the monocytes to dendritic cells I understand that GM-CSF and IL4/3 is required (TNFalpha for mature dendritic cells). For monocyte-derived macrophages I have come across publications which states that the addition of M-CSF to the medium is required. However, there is also some suggestion that incubation with serum is enough for macrophages to differentiate after 5-7days. Is this correct? If I incubate my isolated monocytes with serum (no addition of M-CSF) for a week, what will I actually have? Any help or advice would be greatly appreciated.
Hi! Good question... An adherent monocyte is anyway to be defined as a macrophage. However to get a good differentiation in vitro you need m-csf. The concentration of m-csf is dependent on your purposes and your starting material. Human monocytes isolated from PBMCs can differentiate in macrophages with 10-20 ng/mL. However if you start from vine marrow derived mouse precursor a higher concentration such as 50-100 no/mL might be more appropriate. There are some report claiming that use of higher concentration of m-cad can pre-commit macrophages to acquire an "M2-like" phenotype. That is not happening in my experience though.
For gm-csf you can be more confident in the case of human monocytes. In the case of murine bone marrow derived Mac, as for my experience, it may skew them into a "M1-like" phenotype.
At the end of the day I would suggest you to see how your cells look like in term of phenotype and cytosine production and see whether the basal culture condition you have set are satisfactory for your purposes. A very last consideration: m-csf is one of the most abundant circulating growth factors in the blood. It is a Dropbox factor for monocytes and therefore even a tiny amount in culture, as for my experience again, can only be beneficial. Good luck!!!
Yes, adherent monocytes will differentiate into macrophages. However you will always get better results stimulating cells with either M-CSF (10-20 ng/ml) or low concentrations of GM-CSF (I use 1-5 ng/ml) depending on your needs.
You will get macrophages from adherant moncytes cultured with 10% autologous heat inactivated serum (FCS alone wont cut it). MCSF or GMCSF produces much more consistent differentation.
It is important to identify in any publications how you derived the macrophages, as results can differ between the differentiation methods.
http://www.ncbi.nlm.nih.gov/pubmed/25035950
Also much better viability when growth factors are included - you get alot of death if you culture monocytes for 5-7 days otherwise
Hi! Good question... An adherent monocyte is anyway to be defined as a macrophage. However to get a good differentiation in vitro you need m-csf. The concentration of m-csf is dependent on your purposes and your starting material. Human monocytes isolated from PBMCs can differentiate in macrophages with 10-20 ng/mL. However if you start from vine marrow derived mouse precursor a higher concentration such as 50-100 no/mL might be more appropriate. There are some report claiming that use of higher concentration of m-cad can pre-commit macrophages to acquire an "M2-like" phenotype. That is not happening in my experience though.
For gm-csf you can be more confident in the case of human monocytes. In the case of murine bone marrow derived Mac, as for my experience, it may skew them into a "M1-like" phenotype.
At the end of the day I would suggest you to see how your cells look like in term of phenotype and cytosine production and see whether the basal culture condition you have set are satisfactory for your purposes. A very last consideration: m-csf is one of the most abundant circulating growth factors in the blood. It is a Dropbox factor for monocytes and therefore even a tiny amount in culture, as for my experience again, can only be beneficial. Good luck!!!
It is long known that adhesion itself is capable of inducing monocyte activation and differentiation. You may use M-CSF to accelerate the process, but it is not mandatory. However, depending on the cytokines you use, the differentiation will give rise to distinct macrophage subtypes, as stated by others. I have a paper demonstrating that adhesion per se is capable of activate monocytes without the addition of any cytokines, increasing both CD14, CD16 and monocyte size. You can download it in the following link if you wish. Kind regards!!
https://www.researchgate.net/publication/262415224_Ouabain_inhibits_monocyte_activation_in_vitro_prevention_of_the_proinflammatory_mCD14CD16_subset_appearance_and_cell-size_progression
Article Ouabain inhibits monocyte activation in vitro: Prevention of...
Many thanks for all your fantastic answers! After differentiation I am hoping to stimulate them and measure the resulting cytokine levels. Therefore I am apprehensive about using cytokines during the differentiation process. It seems like to obtain better (and more consistant) differentiation M-CSF should be considered. Thanks again everyone!
Kristina,
nobody has mentioned the use of L-cell conditioned medium so far - which is used instead of M-CSF to differentiate BM-cells into macrophages. I strongly discourage its use, as it contains numerous other cytokines (type I IFNs, IL-6 etc.) which will modulate the phenotype of your cells. Always go with a pure CSF and/or specified FBS (low endotoxin etc.). I highly recommend the following review focusing on macrophage nomenclature and experimental guidelines:
Immunity. 2014 Jul 17;41(1):14-20. doi: 10.1016/j.immuni.2014.06.008.
Macrophage activation and polarization: nomenclature and experimental guidelines.
Best,
Jens
I realize that it's been several months since this thread was active, but I have a follow-up question to this discussion. I realize that most were posting on the benefit of using M-CSF opposed to relying on adherence alone. But, could someone explain if M-CSF is better than using only GM-CSF in medium?
By using only GM-CSF you will get a mixture of macrophages and dendritic cells. If you use M-CSF you will get primarily (or only?) macrophages. I also observed that by using only GM-CSF the differentiation process takes longer than with M-CSF.
I think M-CSF is an excellent differentiation factor from monocytes to macrophage and it works well with 20ng/ml.
"M-CSF exhibits a mostly homeostatic expression pattern, whereas GM-CSF is a product of cells activated during inflammatory or pathologic conditions", means if you are looking for more "ordinary" macrophages then M-CSF is a better option.
Ref. Article Biological role of granulocyte macrophage colony-stimulating...
Hi. I am not an expert of macrophages.
My question is: after 7 days in L929 (murine fibroblast) conditioned medium, do murine bone marrow-derived macrophage precursors differentiate by enhancing expression of Mac-1 and Gr-1?
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