Our Macrophage Generation Media have been developed for the efficient generation of monocyte-derived macrophages (MDM) from freshly isolated peripheral blood monocytes or directly from PBMC as a starting material. The M2-Macrophage Generation Medium XF contains M-CSF and allows for the generation of M2-polarized macrophages. Our M2-Macrophage Generation Medium XF is a ready-to-use medium including cytokines for the directed differentiation of M2-like polarized MDM.
The Macrophage Media XF are serum-free and xeno-free formulations for use with freshly isolated cells. Due to the utilization of exclusively synthetic, recombinant or plant-sourced materials, human serum albumin, purified from human plasma, is the only non-recombinant protein contained in this medium.
All media are optimized for primary human cells. However, we have received feedback from customers that this medium can also be used for murine macrophages.
Figure 1.In vitro macrophage generation with our macrophage media system. Overview of the wide spectrum of macrophage polarization and activation possibilities.
For the M2 Macrophage Generation Medium; it is extremely important that the shelf life of 2 weeks (after addition of the cytokines) is not exceeded; the yield will quickly decrease thereafter. It is best to use the M2 medium as fresh as possible to avoid discrepancies between M1 and M2 yield.
Spinning the cells for 15 min at 350 x g has been proven and tested by PromoCell Research & Development. The QC department uses these settings during testing. Any lower centrifugation value (g-force and/or time) will lead to significant cell loss by means of non-sedimented; but intact; macrophages.
a) We usually perform macrophage differentiation in T75 flasks and 6-well plates. We haven't tested differentiation in smaller formats. But we assume it will be problematic to thoroughly wash the surface of the wells to remove non-adherent cells after the attachment phase. b) Detachment of the mature macrophages is possible but re-attachment can lead to significant cell loss (30-50%).
Please also keep in mind that working in 96/384 well-format has some inherent drawbacks (e.g.; evaporation of media; dry wells; etc.).
Usually; we do not recommend specific plasticware but we have found that with PromoCell’s Dendritic Cell and Macrophage Generation Media; choice of plasticware can have a lot of an influence.
For our M1-/M2 Macrophage Generation Media XF we recommend the Nunc plasticware with Nunclon surface - as not only will the detachment efficiency vary (up to 20%); but also the efficiency of the differentiation process itself may be altered.
Yes the specialized PromoCell media already contain the optimal amount of L-glutamine. Please don't add extra L-glutamine as this can be toxic for the cells.
A concentration > 10% FBS is needed to completely inactivate the trypsin. As most of PromoCell's growth media are serum-reduced or serum-free; the use of a trypsin inhibitor like TNS is highly recommended.
If antibiotics are deemed necessary the recommended final concentrations are:
100 U/ml penicillin + 100 µg/ml streptomycin or
50 µg/ml gentamicin + 50 ng/ml amphotericin B
Please note: Addition of antibiotics can reduce the growth rate of the cells.
After addition of the SupplementMix or SupplementPack to our basal medium do I have to add FCS you obtain the complete growth medium. No further supplementation with serum or growth factors is required. Please note: Our media do not contain antibiotics. If you wish to use antibiotics you can add penicillin/streptomycin or gentamicin/amphotericin B at standard concentrations. Addition of antibiotics can however reduce the doubling time of the cells up to 30-40%.
Yes you can aliquot the SupplementMix upon delivery and freeze down 2 or 4 individual aliquots at -20°C. This way you can prepare smaller volumes (2 x 250 ml or 4 x 125 ml) of complete culture medium and thus extend the time you can use the medium.
Upon arrival the basal media should be stored between 4°C and 8°C the supplements at -20°C.
Please do not freeze our cell culture media. Freezing can lead to irreversible precipitation of media components and the quality can no longer be assured.
The qualitative and quantitative composition of the supplements can be found on our website and in the data sheets of the specialized media. When there is no such information specified; the composition of the supplements is confidential.
The formulation of our basal media is proprietary information. If you need to know the concentration of a particular component for your experiments please contact the PromoCell Technical Customer Service.
It has been shown that phenol red has estrogenic properties. Phenol red-free media are therefore generally used in studies evaluating steroid hormone action in cultured estrogen-responsive cells (Berthois et al. 1986). Furthermore phenol red can also interfere with some analytical methods like photometric analyses.
The Growth Medium Kit allows customization of the end concentrations of growth supplements. It is therefore more flexible than the Medium "ready-to-use" and can eg. be used to prepare a starvation medium.
Please note: Modification of supplement concentrations may have an impact on cell growth. You should test in advance whether and for how long the cells can survive the altered culture conditions.
PromoCell Cell Culture Media "ready-to-use" consist of basal medium and SupplementMix.
PromoCell Culture Media Kits consist of basal medium and SupplementPack.
Addition of the supplements (SupplementMix or SupplementPack; respectively) to the appropriate basal medium will result in identical growth media.
This is not advised. Please seed the freshly isolated CD14-monocytes immediately in the Monocyte Attachment Medium. Adding a culturing step will change the biological characteristics of monocytes very rapidly.
We did not plate the cryo-macrophages in 96-well plates. However we heard from other customers that they have successfully used our macrophages in this kind of plate.
You may find all information regarding the activation and the cytokine concentrations in table 1 (page 5) of our Application Note.
No we do not provide data about the cytokine profile of our M1/M2 macrophages after activation and we do not provide any further data beyond the scope of our quality control.
Even non-activated macrophages do release a certain amount of cytokines. Furthermore you would have to be sure that the release of a certain cytokine is a direct consequence of the activation. Therefore we do not think it is possible to have a general negative control for the cytokine release.
We did not test if the macrophages attach on fibronectin-coated glass.
In general; we recommend activating the cells for 24 hours or at least over night for all kind of activations. If the activation is not optimal in your experimental setting; you can increase or decrease the activation time accordingly.
Unfortunately we did not test this in our hands and it must be tested by the customer. In fact our medium is completely different from RPMI and therefore we cannot predict if this is working. We only know the successful long-term culture from our system with our media.
M1 / M2 polarization also takes seven days in the PromoCell system but the protocol contains two more days for optional macrophage activation. If you only want non-activated M1 / M2 macrophages; the process is usually completed after 7 days.
Nevertheless; PromoCell does not recommend shortening the 10-day protocol because you actually get a plus in viability and cell yield (due to the re-attachment of floating cells) on day 8-10 due to the media change.
The activation of macrophages as such is complete after 24 hrs. However; to maintain the activation status over a longer period of time (i.e.; several days); fresh activation factors should be added with every medium change.
The Cytokine Mix M1 and M2 should not be subjected to further freeze/thaw cycles.
Macrophage polarization: plasticity in health and disease
Macrophages Subtypes: Polarization, Activation and Plasticity
Macrophage cell culture
Human blood cells types
Holotomography-monitored phagocytosis assay of M1 macrophages
Monocyte-derived macrophages: Customized in vitro large-scale differentiation from human PBMC
Standardized culture of assay-ready and fully functional human primary macrophages
Differentiation of M1- or M2-Macrophages from PBMC/Monocytes
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