Dear Xingchen,

we observed exactly the same thing in our lab. We normally differentiate our BMDM with M-CSF (10ng/ml) and we also get around 30 million per mouse THe floating cells are normal. We once tested L-929 and got much more cells, but they did not respond at all after bacterial infection or PAMP stimuli (with expection for cell death).

Until M-CSF was commercially available, the classic approach still in use is BMDM differentiation with M-CSF produced by an L-929 fibroblast cell line in conditioned media. However, critical and often mentioned disadvantages of L929-derived M-CSF are fluctuations in quality and content, which questions the general opinion of equality between M-CSF-derived BMDM and BMDM differentiated with L929 supernatant and the according studies, since not only BMDM preparation but also L292 cell line culturing can highly differ based on the protocols in the laboratories . Moreover, since M-CSF is harvested from the supernatant of a cell culture line, several other molecules released by either living or dead culture cells are isolated too and will come in contact with the differentiating macrophages. Therefore, usage of recombinant M-CSF (rM-CSF) is highly recommended, which is commercially available, to generate BMDMs rather than cell line-conditioned L929 supernatant, which seems in terms of the possibility of a buyable substance with constant quality, i.e. rM-CSF rather archaic and outdated.

All the best and stay healthy,

Marc

Dear Marc Herb,

Thank you for your detailed answer and it really helps!

I totally agree with you that the L929 supernatant contains m-CSF as well as other unknown factors, however, I indeed never encountered any issue with the L929 conditional medium. The BMDMs are responding, at least, to inflammasome activators very well. But I am not an expert in terms of bacteria infection on BMDMs, so maybe there are some issues as you mentioned.

I am wondering if you can provide a lit bit of details of your protocol by answering the following questions?

1), How many plates you seed for BMDM differentiation for one mouse? I used to do 10 plates with L929 medium.

2), On day 3 or 4, do you replace with fresh medium containing m-CSF? Will this damage the yield of total BMDMs since many floating cells are removed.

3), Now I am using the tissue culture plate instead of the petri dish since the BMDMs (with m-CSF) barely attach to the petri dish from BioDot (we currently have in our lab). It attaches very well to the petri dish from Fisher Scientific (the one I used from my former lab with L929 conditional medium). Do you have any comments on the plates you used?

Thank you in advance!

Best.

Xingchen

Dear Xingchen Dong,

For M-CSF medium we use 6 petri dishes per mouse. The cells, which differentiated to macrophages will sit for sure. The rest of the floating cells are ,accordingly, not macrophages, so you do not want them to sit.

No, at day 4 we add another 4 ml of fresh medium to each plate but do not replace it, because at day 4 most of the cells did not yet differentiate and do not sit. So by removing your medium you will loose a lot of potentially differentiating macrophages.

For a very detaled protocol how to isolate and cultivate BMDM with M-CSF please have a look at our methods paper:

Article Highly Efficient Transfection of Primary Macrophages with In...

If you have any further questions, please feel free to ask any time.

All the best,

Marc

Dear Xingchen Dong

I think L929 conditional medium is quite differ from labs. When my teacher pursued her doctor's degree, she can yield around 5 dishs of 20cm BMDMs per mouse. The L929 Medium she used in made after culturing 3 weeks until the color totally turned yellow. She also told me using L929 is better than M-CSF.

But I can't reproduce her experiment. My l929 will die after 10 days and I can only get 15 millon BMDMs per mouse with lots of bublys.

Could you tell me the protocol of production of L929 Conditioned Medium in your lab? May be the key to produce more BMDMs is in the L929 medium itself.

Dear Junyan Zhou That's so true that the quality of L929 medium varies depends on who prepared it. We culture the L929 cells in the 2L cylinder container and put it on a rotor in the incubator. In this way, we avoid overgrowth and cell death as growing in the dish. We collect them after 3 weeks, and the medium was not totally turned yellow. Maybe you should change your seeding density since 3 weeks is a pretty long time for cell culturing.

Dear Xingchen Dong

Wow, It appears that we use the same way to produce L929 medium. My teacher also told me use the cylinder container and rotor. She said she learned it in Tsinghua University

But unfortunately she forgot the seeding density.Thanks for your advice and I think you're right, I used to seed 2 L929 dishes with high growth rate in the 1L cylinder container, which has a suface area that is about 6-9 times of a 10 cm dich . I will follow your advice adjusting the seeding density, could you please tell me the seeding density in detail？And do I need to supplement with some Glutamine after a while？My L929 will died in the roller bottle before the medium turn yellow.

Hi Junyan Zhou ,

I don't really remember the exact seeding density since one preparation of L929 could last a couple of years. I left my previous lab, and never prepare it later. But roughly, we seeded, if correctly, 3x confluent 15cm dishes of L929 cells into a 2L cylinder with a total 1L medium in it.

The medium formula is the same as BMDM culturing medium (expect the L929):

10% FBS

1% Pen/Strep

1% HEPES

1% L-Glutamine

DMEM/F12 medium

We could observe lumps (probably dead cells) in the cylinder at day 21, and we simply centrifuge and filter it.

I think medium turns yellow is normal, and I am wondering if your low yield of BMDMs is due to your bone marrow isolation technique? One key thing is to add fresh L929-containing medium after 3-4 days of BMDMs differentiation.

Dear Xingchen Dong

Thanks for your detailed answer which helps me a lot in L929 culture. Your old friend said you are a good person LOL. You can guess who she is