Other growth factors participate, especially IL-3, GM-CSF, Flt3L. L929 cells make many cytokines and growth factors. MCSF also comes in several isoforms that can have differential effects. Rajavashisth and Lusis are working on this.

I have been using M-CSF from peprotech in macrophage differentiation for many years for macrophages from both humans and macaques, and it seems to work pretty well. But I have no experience with L929 media, so I cannot directly compare.

But there are some other things that might be interfering with the maturation of your macrophages using normal media. One could be the way the M-CSF is added to the media, because if you make up a large volume of media, say 500 mL and add M-CSF to it and store it at 4°C, the M-CSF will break down because it is much less stable at lower concentrations. I use it at 10 ng/mL final concentration, but store it at 100 ug/mL in water and add it to media each day before I feed my cells.

In the past I have tested different amounts of M-CSF and have seen that the amount of M-CSF one uses can influence maturation. We found that using M-CSF at 2 and 5 ng/mL was less optimal than at 10 ng/mL, but that going above 10 ng/mL to 20 or 50 did not make much of a difference.

Also, keep in mind that the culture dish you use to grow the macrophages (glass or different types of plastic) may alter their activation and maturation, as can the donor from which you derive the cells if you are looking at primary cells.

I was in a lab that would generate macrophage cell lines from different tissue (spleen and brain) that were all growth factor dependent. The PI in the lab used a cell line that he had generated earlier called LADMAC as a source of M-CSF (available from ATCC). We found it to be a better source of M-CSF than L929 (Especially when growing macrophages from bone marrow. With using L929 and LADMAC it is probably other factors in the supernatant besides the M-CSF which helps in the generation of macrophages. We would test each batch of media for ability to generate macrophages from bone marrow as there could be some variability in amount of M-CSF present.

If you just want to have enough macrophages for functional experiment, L929 CM is a better choice (no reviewer will challenge that). However, if you are investigating macrophage differentiation, defined medium is critical, so use M-CSF instead. Other cytokine cocktails are also literauted for differentiating myeloid cells, such as SCF+IL-3+IL-6.

We have only used L929 CM and had bone marrow derived macrophages that are nearly 95-98% 5 pure. They also respond well to TLR stimulation. We have never used recombinant M-CSF. I agree that for asking questions about macrophage differentiation it may be good to try recombinant cytokine.s

I often think of Conditioned media as being "chicken soup" with no two being alike. While you can look at amount of M-CSF produced by cell lines, it is probably the other factors that are produced by cell lines that help in macrophage development.

Our main focus is neutrophils but also work on mouse macrophages. We use conditioned medium from a cell line called CMG14-12 (original reference: http://www.ncbi.nlm.nih.gov/pubmed/10934646) which we originally received from Eric Brown at UCSF/Wash U. People say that it contains much more M-CSF than L-cells but we have never directly compared. 10% CMG-conditioned medium triggers robust macrophage development in our hands.

Dear Mustafa,

I've been differentiating macrophages and dendritic cells from mouse bone marrow progenitors for some years now. For DCs I've started to use rGM-CSF and then changed for the supernatant of J558 that produce this growth factor. After some adjustments in supernatant concentration, the expansion and purity of DCs was the same as I was getting with the rGM-CSF (95-98%) and it is much cheaper. For macrophages, I've only worked with LCCM (supernatant of L929 that produce M-CSF). Again, you need to titrate the volume to add to the culture for each lot of supernatant you produce but you'll get 92-95% pure macrophages. On the contrary to DCs, macrophages will not expand, so you'll have the same number of differentiated cells as bone marrow progenitors.

Thanks for all the answers. I am looking at 2 aspects:

1) Inflammatory (TLR) response in macrophages (BMDM)

2) Macrophage differentiation

- When looking at 1, I often realize that when I use recombinant MCSF I get more IL6 production, for instance. But at the same time my macrophages look a bit less confluent. However when I use L929media they get super confluent and out of 1 animal I end up with 40 million cells on average. Also for certain non-coding rna I donot see any expression when using L929 (maybe because basal levels go up ?) I am starting to doubt if recombinant-mcsf actually works. They may very well be differentiating into monocyte/macrophage intermediate. I have to do facs to be sure i recon.

By switching labs I switched from L929-CM (freshly added every 3 days for one week) to recM-CSF (Peprotech, one dose for the whole week without refreshing medium) and I now yield half the number of macrophages but they seem to be more homogeneous and more "naive" than L929-macrophages, which is quite useful when measuring small changes in gene expression caused by the not-very-drastic phenotype changes we look at. However, as you're looking at TLR responses, I guess you can expect stronger changes in gene expression and you might not need the subtlety of the M-CSF-macrophages and just go for the higher yield?

For differentiation experiments I would definitely choose the recM-CSF as you have no idea of what you're adding to your cells with L929-CM...

I have used both macrophages derived from both rMCSF and L-cell conditioned media to study responses to M. bovis. I found that L-cell derived macrophages gave higher cytokine secretion than rMCSF derived macrophages. Using the L-cell dervied macrophages enabled me to detect cytokine release at lower MOIs and earlier time points. We also thought this was due the rMCSF macrophages being in a more immature state.

Richard Stanley answered all these question in his papers from the late 70s and early 80s. I used to think like  Pieter Goossens, that we don't know what's in L929 and that there are other growth factors, etc. (basically that L929 is really dirty). and that's not true at all. Read Stanley, particularly anything where he's the 1st author. He's the best at MCSF/CSF1R/Macrophages in general

hello , what is the concentration of  M-CSF released by L-929 in condition media of this cells?Do you know about this please guide me

your sincerely

Do you do a selection before harvesting the supernatant from L929?

Which media you use to cultive L929? AlphaMEM or DMEM? And to stock?

Thank you very much. 

how can i measure m-csf concentration in L929 condition media (complete DMEM with 15% L929 supernatant)? and what is the recommended concentration ? 

Hi Dr DG. I don't think a lot of labs are measuring the M-CSF concentration in their L929-conditioned media. You just compare the yield and phenotype (CD11b - F4/80 FACS) with different L929-CM concentrations (and you can take your standard rec M-CSF condition along if you want, but the yield should at least be higher with the L929-CM), and you empirically determine your desired L929-CM concentration, which could differ from batch to batch. Go for the lowest concentration possible that still gives you both a good yield and a high level of differentiation (typically between 5 and 20%).

I would add: I think the point being missed here is: L929sup isn't intended to be used for experiments!!

Use recombinant MCSF for these!!

Use L929 for the regular culture of the BMDMs & propogation of the progenitor population :)

Use recombinant MCSF for controlled experimental conditons :)

...hope this helps!

--Luke

 Pieter, and Luke: thank u so  much, it does really help !

I have another question: what is the best way to count the macrophages ? 

To count them you'll have to detach them first I'm afraid. But then you could use whatever method you prefer (counting chamber, automatic cell counter,...). After 7 days of culture, all the non-macrophage cells have died so there's no risk of overestimating the macrophage numbers.

I'm guessing (since your asking the question) your thinking of using something other than a traditional hemocytometer. I'm sure there are other ways (e.g. automatic cell counters). Whatever you do (& this kind of goes for all experiments actually) make sure you PRACTICE & can get correct/accurate consistently!

Your probably thinking...well...duh. But the way I got this issue straight (because it is a big issue--other people have tried to count and plate my cells & is been disastrous!) was working with the 1 cell line I do have,(I don't use it on a regular basis, but I have freezes) it's the very popular murine monocyte RAW cells. These cells are like HeLa cells--very hard to kill or slow down, easily transfectable, basically you can do whatever you want with them (the catch is, as with any cell-line, it's probably not biological relevant! haha).

...anyways they are a adherent monocyte cell that's more similar than different to the primary BMDMs I work with everyday. You could use BMDMs to practice, but I didn't want to waste them initially.

What you NEED to do is set aside a day or two and start with a set number of cells by FACS sorting (e.g. a million) then take them back to lab and see how many you count AND count it again and again until you get at least 3-5  similar readings. I'll bet you 5cents;) that you'll lose at least 10% of your cells initially. Where are they?!?! in the tubes and pipet tips! Remember--MACɸS ARE NOT JUST "REALLY" MOTILE....they are THE MOST MOTILE (& STICKY) of all cells--& I think this is why people struggle to count & plate them. Because when you count and plate, the macɸs are inside the tube or pipette frantically grabbing anything they can get a hold of. So, if your times vary in the counting/plating, it's no surprise the % of macɸs that are lost (i.e. stuck to tissue culture ware) varies as well.

So what can you do about it!?!?!? haha--GOOD QUESTION!

Well, I would say try to be consistent with the experimental groups as much as possible (again--duh, right!), also I look at my cells constantly under the light microscope! (I know everybody says that) At the end of the day, if you look briefly at 2 surfaces with the "same number of cells"  at 4x (& even 10x)--THEY SHOULD LOOK APPROX. THE SAME!! so if I'm doing something where plating number is critical, I'll regularly check, because they density should be approx. the same. This ability to really truly "see if the plating numbers are right" comes from experience & doing it a lot (which doesn't really help you--but it's the way it is--& the truth).

HONESTLY, I avoid experiments that require precise cell plating numbers (such as cytokine secretion ELISAs) as much as possible.

The best example I have of this straegy is for assessing macrophage proliferation. The classic assay is thymidine incorporation--which basically measures the amount of DNA in each well--you can image if your initial plating is slightly off...yeah...end results MIGHT be off ;)

SO I DON"T DO THAT ASSAY for proliferation!!!!!!

I do CFSE dye dilution now which is not dependent of initial numbers and plating of groups to be highly accurate because there is a time "0" reading that sets ta relative baseline for each group. I won't get into anymore, but basically--look for experiments that don't require perfect plating (& yes, I know, sometimes it can't be avoided!)

BOTTOM LINE: their is no magic fix! sorry, that's what you get for working with primary BMDMs. work with HeLa cells if you can't handle it.... haha, jk!....I wouldn't wish that on anyone!

If there is a "trick", I think it's to avoid experiments where plating number is very sensitive. ALWAYS KEEP IN MIND & BE THINKING ABOUT THE FACT THAT THESE CELLS ARE THE MOST MOTILE & STICKY CELLS ON THE PLANET ;)

 should i use trypsin to detach the cells ? 

No, never detach BMDMs using trypsin. There's plenty of different methods people use and that have been discussed on this website (e.g. https://www.researchgate.net/post/What_is_the_best_way_to_harvest_adherent_macrophages_out_of_48-well_plates ), but trypsin is not an option. My favourit is PBS + EDTA + Lidocain, but I'm sure we won't reach unanimity on this...

 I use to get macrophages from bone marrow and after 5-6 days growing I always detach with trypsin and never had a problem with that... maybe other type of macrophage i can´t use trypsin as the answer above, but I am not sure...

now i am staining peritoneal macrophages for flowcytometry , i use fcblocker, f4/80, and cd11b. I read some protocols but since i have no previous experience with flowcytometry, can u give me simple protocol or guidelines for that purpose especially how to adjust antibody concentration for flowcytometry?

We use scrapper for cell detaching after 7days of treatment with M-CSF. We didnt encounter any problem

We use also L929 derived m-Csf. May be one big difference is the possibility to express from the gene all the other m-csff mRNA variants and so the different forms of m-csf? I never look at the exact cytokine/chemokine compsition, but may be there is also Il6... If someone can access a multiplexed ELISA (or RT QPCR), it could helps!

I've always used L929 but am thinking about switching to M-CSF as I'm doing some phenotype switching experiments. Just a suggestion for counting etc - I grow these cells in teflon bags that I source from the USA (clean room prepared but I autoclave them pre-use as they're not technically cell culture bags - but are a LOT cheaper). It means that counting is a doddle because they don't adhere, and they are very naive because you don't have to scrape them or traumatise them in any way to get them out. A quick shake and they all just drop into solution. If anyone wants company details I'll let you know where I get them from!

Charly Oakley · - I'm very interested in the teflon bags for BMDM culture, can you send this information to me? julie.schwartz(at)Mcgill(dot)ca

Also, I recently read that in a comparison of BMDMs generated using L929-conditioned media versus recombinent M-CSF, there were some differences in signaling & activation.  I don't have the reference on hand at the moment, so take that with a grain of salt, but it's something to consider/be aware of for our experiments. 

Dear Dr. Julie,

Could you please send me the details protocol for the growing of L929 cells to harvest medium, which will use to differentiate bone marrow macrophage.

Regards,

Karan

If you have an efficient and already proven L-929 cell culture that produce enough quantity M-CSF it is better to use its supernatant instead of recombinant M-CSF. There are other components in L929 SN (not only M-CSF) that have positive effect on BMDMs differentiation.

Thanks!!