Usually people add M-CSF or L929 conditioned medium to regular medium (e.g. DMEM + 10% FBS) to differentiate bone marrow into BMDM (bone marrow derived macrophages) for 7 days.
I have an argument with my friends about the use of M-CSF (or L929 medium ) in the following cell treatments.
Do you use regular medium (DMEM or RPMI + 10% FBS) in the following BMDM treatments or regular medium plus M-CSF (or L929 conditioned medium)? We found the curves are different with different medium.
Thank you so much for any input.
I differentiate my cells using 30% of L929 supernatant for 7 days (adding extra medium on day 4). After differentiation I stimulate my cells in medium containing 5% of L929 supernatant.
Thanks, Andre. Could you please let me know why you use L929 supernatant instead of regular medium (DMEM + FBS)?
Xianfeng, I don't use pure LCCM (L929 supernatant). I acuatly use (for differentiation) what it's called R20/30.
This means
- 20% FBS,
- 30% LCCM (L929 spn)
- 50% DMEM/RPMI
During my experiments I use a more diluted composition called R10/5,
- 10% FBS
- 5% LCCM (L929 spn)
- 85% DMEM/RPMI
Pure LCCM is acidic because of the long time it takes to produce. If you use it pure it will kill your BM cells before they differenciate.
Andre, I totally understand you. People argue that we differentiate BM cells into BMDM with M-CSF(or L929 conditioned medium) containing medium for 7 days, and that the cells are macrophages after differentiation and we dont need to add M-CSF or L929 medium into DMEM+FBS when treat cells(macrophages) with some a drug. Hope I describe clearly.
What do you think? Thanks for any idea.
I think for the following cell treatments there is no logic behind using mcsf or l929 conditioned media. Because after 7 days your cells are already terminally differentiated. Infact, it is better not to use, because the effects you see from your treatments may be unnecessarily influenced by ingredients of the conditioned media. Anyway, I use just DMEM+10% FBS for my treatments post 7 days, and they work fine.
Hi Sreeram, thanks for your input. When you differentiate cells you have to add M-CSF or L929 conditioned medium. And then you withdraw M-CSF (or L929 conditioned medium), no any effect of this "withdraw" on cells? I think the final responses from cells are drug effect plus "M-CSF withdraw" effect.
My cells always suffered if I removed LCCM during my stimulus. The protocol I use is followed by the Zambonni group and it was pubblished at Nature Medicine.
Hi Andre, thank you for the information. I found it. http://abc.org.br/IMG/pdf/doc-4272.pdf
Yes Xianfeng
The L929 cell and the protocol I use came from Dr Zamboni's lab. I've always had at least 95% F4/80pos cells. As for any differences during cell stimulation if you use R10/5 for all your tests (unstimulated) you won't have any problem.
The addition of L-929 cell culture supernatant (10-30%) and M-CSF (10-100 ng/ml) is essential and always gives better results in comparison to L-929 SN / M-CSF free media. However, if you add more quantity of L929 supernatant (which also contains M-CSF) it is better to reduce the quantity of supplemented M-CSF.
You can use either L929 cells media or M-CSF, in both cases you treat the cells for 7 days. After this time, the cells are supposed to be differentiated already, that is why you do not need to add additional M-CSF/L929 cells media in the following treatments.
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