I am not sure about some of the components in your medium. There are many well known media (e.g. L929 medium). Why don’t you try them? The addition of L-929 cell culture supernatant (10-30%) is essential in your case.

1. We usually add 0.05 mM ß-mercaptoethanol and it has good effect on our BMDMs.

2. M-CSF concentrations could vary between 10 and 100 ng/ml. However, you should be careful if you add L929 supernatant because it also contains M-CSF.

3. You can change the media (or add extra medium) after 2-4 days of incubation. Probably you will have confluent plates after 7 days of incubation.

For M1 activation, you can try with IMDM containing 10% FBS and 100 ng/ml LPS or 100 ng/ml LPS with 50 ng/ml IFNγ; for M2 activation, you can use IMDM containing 10% FBS with 10 ng/ml IL-4 and/or 10 ng/ml IL-13.

For more detailed information I would recommend reading:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3728835/

Thanks for the answer! I have one more question - how long do you stimulate BMDMs for M1 and M2? 24 or 48h?

This is a bit of a long winded answer so bear with me. As with most science unfortunately, this topic is very subjective, so you must start by asking objective questions:

So, before using culture media, we should ask: Why are we using that component?

So:

· 4500 mg/L glucose

This is very high glucose, 5 times more than physiological levels. If you can I would reduce this to 5mM (roughly 900g/L). Otherwise you are culturing in over diabetic levels.

· L-glutamine 4.0 mM

This is also very high but i'm not sure what any consequences would be, so keep it at this level, most people use 2mM. This is a major fuel for macrophages.

· 10% Foetal Calf Serum HyClone

I still don't think we're quite ready to drop serum from media yet, but there is a big case for not using it, it's from a different animal and we really don't know what's in it (batch dependent, location dependent).

· 20 ng/ml MCSF

This is essential for macrophage growth and survival in many physiological contexts, it works in concert with GM-CSF and other factors to control cell phenotype and renewal. This can be supplanted by IL34 in some tissues. Though for most studies this is a clean way of getting a 'pure' macrophage culture.

· 100U/ml penicillin & 100mg/ml Streptomycin

Peace of mind when culturing.

· 1 x Non Essential Amino Acid

These are usually in DMEM and serum anyway.

· 1mM Sodium Pyruvate

I never understood this one, it is absolutely unnecessary in almost all cultures. Cells use glycolysis to get this, if you skip this step, then you will change the way a cell is metabolically activated. We don't know the full consequences of this yet, but I imagine this could impact the pentose phosphate pathway and the way cells generate ATP from glycolysis.

· 0.01-0.05 mM 2-Mercaptoethanol

Again, I've never really understood this. The line used to be cystine to cysteine conversion, especially for T-cells which lack the enzymes. But we have found this to be absolutely unnecessary, all the immune cells we use don't need this.

For M-CSF concentrations, this is vital for cell growth survival, so it should always be there, cell will die without it (especially if they are addicted to it after one or two treatments), but if you add it too regularly, you will kick in more cell cycles and shorten the life of the cells, but mature them faster. For mice, I would recommend feeding M-CSF 10ng/ml on day 0 and day 3, replace media with fresh media + MCSF on day 4, feed MCSF day 6, experiment day 7. (seed cells at 180,000 per cm2 of culture area)

For human cells, the monocytes do not like differentiating completely in M-CSF alone, but if you use 50ng/ml and feed them every day, they will be better (but this is expensive). The seeding density in my view is better very high 200,000 per cm2 of culture area).

I would not recommend L-929 media, because you really don't know what is in it and this can be variable in different labs and days. L929 also has GM-CSF, so it will give more cell heterogeneity, which is a bad thing if you are doing controlled experiments. The advantage of L929 is that it is cheap and after all, physiological tissues will have low levels of tonic GMCSF and high levels of MCSF (depends on the tissue), so you may be getting a sense of real heterogeny.

GMCSF alone or in combination with MCSF can be good to use if you control what you're looking at. I have alway envisioned macrophage biology moving into MCSF+GMCSF cultures as a standard.

Macrophage polarization is going out of fashion these days, but following Dimitar's concentrations will get you there (though it is unnecessary to change the media type). The problem with polarization is what question you are asking and what you are actually looking at. If you look 2-4 hours post treatment you will see the gene effects of the primary stimulation, after 8 hours this will be secondary and by 24 hours the cells have gone through a multitude of signalling cascades. Specifically, for M1, this will also involve nitric oxide shutdown of mitochondrial metabolism and glycolytic compensation. Cells never really become polarized in vivo, but they can share characteristics with some TLR/ interleukin stimulated conditions we see in vitro.

We really need more study to improve decade old tissue culture techniques. And to be objective we should know the environment of the cells we are trying to culture. So for monocytes, we should really be using 100% human serum, because that is where they live. But this would be too expensive for most labs, so we've decided to create an even more artificial environment. We must unfortunately compromise.

Thanks so much for the very thorough recommendations Luke C Davies !

Just wondering what your thoughts were on seeding density recommendations, as the numbers area all over the place as one reads the literature (if the density is even provided; some folks just plate everything that comes out of the bone marrow, without even indicating which/how many bones were flushed!).

This article finds that a low seeding density of 7-10E3 cells/cm^2 provides a higher quality cell yield (see paper below).

Article Cell density during differentiation can alter the phenotype ...

Ernesto Pena you bring a valid issue. Seeding density! In my hands contact inhibition dogma isn't really an issue unless the cells are really dense, for me as can be seen with THP-1 cells, it is not the density per cm2 that is important, but per ml, which means the media acidity, soluble factors or sheer lack of access to nutrients are the growth inhibitors rather than contact. This makes sense as cells grow extremely dense in vivo (as long as they are well nourished and oxidated). We aren't growing epithelial cells!

The paper you show is intriguing though, it highlights something we know and see in all our macrophage cultures (the size heterogeneity), the problem is: what is more physiological? Yes, a big cell is usually associated with macrophages, but macrophages taken from in vivo (mouse peritoneal macrophages for instance) are tiny, even smaller than cells such as undifferentiated human THP-1. The issue is we are trying to match in vitro culture conditions to perfectly emulate old in vitro culture conditions rather than asking what the in vivo physiological environment is. As long as we can make an argument for what we did when we publish it doesn't matter... But I say it does, I would take progress over tradition every time and tire over the same rookie excuse of this is what Blabla et al. did in 1980, therefore it is correct.