Abstract
Bone marrow derived macrophages are a type of white blood cell that can be isolated from mammalian bone marrow. In this protocol, a method is described in which bone marrow cells are isolated from mouse leg bones (femur and tibia), and then differentiated to bone marrow-derived macrophages in approximately 10-day culture using L929-conditioned medium.
Keywords: Isolation, Culture, Bone marrow-derived macrophages
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. This protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA [Chen et al. (unpublished)].
References
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Sigma.Macrophages could attach to the dish very tightly, you'd better to add Trypsin-EDTA to the dish and incubate for 2 min to loose the macrophages before using cell lifters.
Macrophages usually attach to the plastic firmly while other cells, e.g. fibroblast cells, loosely attach. Thereby, other cells can be washed out by using PBS during the culture and after about 10 days, almost all cells become attached macrophages. You can also use Trypsin-EDTA to facilitate the process... Macrophages can still remain attached to the plastic after 1min treatment with Trypsin-EDTA while other cells become completely detached ...
Usually one time use.
1. You don't need to distinguish the MSCs and the hematopoietic stem cells because only the hemnatopoietic stem cells can be induced by the L-conditioned medium which contains M-CSF and then attached to the plastic firmly. All other cells be lost during the medium-changing/washing process.2. The scrape won't hurt the cells. The cells can be cultured and used for at least two weeks.
Never tested the condition without FBS. Not sure about this.
That will definitely affect the living of the BMM. If the L929 cells are not in good conditions and you still want to use the conditioned medium of this not-good conditions, you can try to increase the amount of the conditioned medium in the BMM medium to 20%-30%.
Initially at 2 bones/10-cm dish and the cell density is around 10^4/ml.We usually directly use cell lifters to scrape the the macrophages since the cells are very sticky to the plastic.
Hi! Ran, I used LPS to stimulate the mature BMDM growed in 150cm dishes for my CHIP analysis ,but I did find that gene of inflammation were inhibited strongly ,despite the mRNA was upregulated. RPMI 1640 with 10% FBS was used in my stimulation stage. Look forward for your reply.
With the protocol, you won't get significant contamination of erythrocytes or bone fragments. You don't need to eliminate the erythrocytes or bone fragments immidiately --- those residual erythrocytes or bone fragments will be washed out when you change the media after the BMM cells are attached. 50oC to inactivate some unknown cell inbitors in the FBS.
thank you for your suggestions.The culture cells are better than last time.Here is a question: could BMM`phi` growth medium be used for recycle? I worry about the concentration of M-CSF.
No. I don't suggest to recycle the BMM growth medium. You can use the L929-conditioned medium (L-cell conditioned medium) instead of M-CSF to lower the cost. Actually, the L929-conditioned medium is used in this protocol.-- by Ran Chen
Thank you very much. Here is my email address:yuxiongbao8@gmail.com I wish we could exchange experience later.
The purpose is to sterilize the bones, otherwise, bacterial contamination usually occurs. It wouldn't fix the marrow.
Yes, you can activate the cells with LPS.
Use 20 ng/ml M-CSF instead of L-conditioned medium.
L929 conditioned medium is an alternative to M-CSF. The protocol above is to get as large number as you can. If you want highly pure macrophages, following the step below may help:During the culture of BMM cells, wash cells twice with PBS every 2 days, and then change to fresh BMM growth medium. -- Macrophage progenitors adhere to the plastic firmly and are not washed away. Fully differentiated macrophages can be harvested and used at day 7.
Hi ! I gotta a question about Procedure 3(g), how could you eliminate erythrocyte and fragments of bones? I usually use red cell lysis buffer as well as filter with 200 nylon mesh , but the rate of adhesion is very low . I am confused about that.