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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.

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Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMM’phi’)

Immunology > Immune cell isolation > Maintenance and differentiation
Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
Vol 2, Iss 3, 2/5/2012, 33579 views, 15 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.68

[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

  1. L929 cells
  2. RPMI 1640 medium (RPMI) (Life Technologies, InvitrogenTM, catalog number: 11875-093)
  3. Fetal bovine serum (FBS) (Atlanta Biologicals, catalog number: S10350)
  4. Stock penicillin/streptomycin (P/S) (Life Technologies, InvitrogenTM, catalog number: 15140-122)
  5. DPBS (Life Technologies, InvitrogenTM, catalog number: 14190-250)
  6. 75% ethanol
  7. Bone marrow growth medium (see Recipes)
  8. BMM’phi’ growth medium (see Recipes)

Equipments

  1. Cell culture incubator
  2. 0.22 μm filter
  3. 27 g needle and 1 ml syringe
  4. Scissors and forceps
  5. 15 ml cell culture dish

Procedure

  1. Preparation of L-cell conditioned medium: culture L929 cells with initial 50% confluence in RPMI + 10% FBS for 5 days, collect the medium and filter with 0.22 μm filter, aliquot and store at -20 °C.
    Note: Incubate FBS at 50 °C for 30 min before using.
  2. Prepare bone marrow growth medium and BMM’phi’ growth medium (see Recipes).
  3. Isolation of mouse bone marrow cells.
    1. Sacrifice mouse and immerse mouse in 75% ethanol.
    2. Clip the skin mid-back and remove the skin from the lower part of the body.
    3. Remove tissue from legs with scissors and dissect away from body.
    4. Clean remaining tissue from the pelvic and femoral bones and separate at knee joint. Be careful not to break the bones. It is important to make sure that all the tissue is removed from the bones since cells associated with this can contaminate the marrow preparation and potentially overgrow the macrophages.
    5. Immerse the bones in 75% ethanol for 5 min, then immerse them in DPBS for 5 min and leave them in RPMI+P/S until the next step.
    6. Cut off each end of bone.
    7. Using a 27 g needle/1 ml syringe filled with bone marrow growth medium; expel the bone marrow from both ends of the bone with a jet of medium directed into a 15 ml cell culture dish.
    8. Change medium every 3 days. While in culture, some of the cells become attached, while many of them still grow in suspension, so spin-down and re-culture those cells in new dishes.
    9. After about 10 days, almost all cells become attached BMM’phi’s, and then BMM’phi’ growth medium is used for further culture and tests.

Recipes

  1. Bone marrow growth medium
    1. Add 5 ml of P/S to a 500 ml bottle of RPMI. Remove 100 ml and save in a clean 500 ml bottle.
    2. Add 20 ml of heat-inactivated fetal calf serum and 40 ml of L-cell conditioned medium, and mix well.
  2. BMM’phi’ growth medium
    RPMI+10% FBS+10% L-cell conditioned medium

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

  1. Gordon, S. (2003). Alternative activation of macrophages. Nat Rev Immunol 3(1): 23-35.


How to cite this protocol: Chen, R. (2012). Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMM’phi’). Bio-protocol 2(3): e68. DOI: 10.21769/BioProtoc.68; Full Text



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3/3/2016 6:57:59 PM  

Tariq HUssain
China Agricultural University, Beijing

Hi,
I have the same question, that how I can purify only macrophages from other cells of the bone marrow.

3/6/2016 11:28:01 PM  

Ran Chen (Author)
Department of Genetics,Stanford University

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 ...

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8/28/2015 8:37:55 AM  

P Singh
Wayne State University

Thank you Dr Chen for this protocol. Can we subculture and passage 1-2 time these differentiated macrophages or are they just one time use following differentiation?
Many thanks
PS

9/1/2015 6:04:29 PM  

Ran Chen (Author)
Department of Genetics,Stanford University

Usually one time use.

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2/3/2015 7:37:19 PM  

zheng PM
Xinhua Hospital

Hi, thank you for your protocol. Now I'll try to do the same exp, and I have some questions.First, how did you distinguish the MSCs(marrow mesenchymal stem cells)and hematopoietic stem cell, due to the MSCs also easy to adhere to the plastic dish.If the MSCs can also be induced to Macrophage after stimulated? Second,you said you usually directly use cell lifters to scrape the the macrophages since the cells are very sticky to the plastic, I wonder whether this will affect the cell state, and how long these detached cell should be cultured, because I'll use these macrophage to do next exp. Thank you for your kindly help.

3/3/2015 1:29:55 AM  

Ran Chen (Author)
Department of Genetics,Stanford University

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.

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2/3/2015 7:32:47 PM  

Hi, thank you for your protocol. Now I'll try to do the same exp, and I have some questions.First, how did you distinguish the MSCs(marrow mesenchymal stem cells)and hematopoietic stem cell, due to the MSCs also easy to adhere to the plastic dish.If the MSCs can also be induced to Macrophage after stimulated? Second,you said you usually directly use cell lifters to scrape the the macrophages since the cells are very sticky to the plastic, I wonder whether this will affect the cell state, and how long these detached cell should be cultured, because I'll use these macrophage to do next exp. Thank you for your kindly help.

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9/8/2014 5:55:32 PM  

yu liming
Nanjing Medical University

Preparation of L-cell conditioned medium: culture L929 cells with initial 50% confluence in RPMI + 10% FBS for 5 days , but others use RPMI without FBS for 7 days, does it matter?

9/12/2014 10:52:35 PM  

Ran Chen (Author)
Department of Genetics,Stanford University

Never tested the condition without FBS. Not sure about this.

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4/5/2014 5:48:09 AM  

khlj

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4/5/2014 5:47:47 AM  

khlj

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10/4/2013 8:10:52 AM  

chunxiao hu
pharmocology of UIC

Thank you very much for sharing the protocol. If the L929 cells cultivated in the same medium for 5 days, the cell condition is not very good. Will this influnce the living of the BMM. And how to get L-cell conditioned medium of high quality?

10/5/2013 11:40:22 PM  

Ran Chen (Author)
Department of Genetics,Stanford University

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%.

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9/10/2013 3:10:23 AM  

Arturo Wilkins
UNAM

Hi, I would like to know how many plates do you seed and at what cell density. Also, how do you dettach the macrophages once differentiated.

9/10/2013 8:44:35 PM  

Ran Chen (Author)
Department of Genetics,Stanford University

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.

7/2/2014 10:39:35 PM  

yu liming
Nanjing Medical University

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.

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9/4/2013 3:40:56 AM  

yu liming
Nanjing Medical University

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 . what`s the purpose of incubating FBS at 50 °C for 30 min before using.

9/6/2013 10:14:42 PM  

Ran Chen (Author)
Department of Genetics,Stanford University

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.

9/7/2013 6:27:12 PM  

yu liming
Nanjing Medical University

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.

9/9/2013 12:46:24 AM  

Ran Chen (Author)
Department of Genetics,Stanford University

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

9/9/2013 2:02:00 AM  

yu liming
Nanjing Medical University

Thank you very much. Here is my email address:yuxiongbao8@gmail.com I wish we could exchange experience later.

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2/12/2013 8:54:29 AM  

What is the purpose of immersing bones in ethanol? wouldn't this fix the marrow?

2/14/2013 3:08:40 PM  

RAN CHEN
Stanford

The purpose is to sterilize the bones, otherwise, bacterial contamination usually occurs. It wouldn't fix the marrow.

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1/11/2013 12:41:35 AM  

Hi thank you for the protocol

I was wondering if i can activate the cells i would have isolated with a substance like lipopolysaccharide?

1/14/2013 4:39:40 PM  

RAN CHEN
Stanford

Yes, you can activate the cells with LPS.

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11/16/2012 9:49:43 AM  

Intelligence and simplicity - easy to unedrsatnd how you think.

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4/11/2012 10:12:28 PM  

Hi,thanks for your sharing!
i really want to know more details about the procedure if i use the M-CSF to the culture of BMDM,such as the exact concentration of M-CSF, when to change the cutlure and so on.

4/14/2012 1:54:25 PM  

Ran Chen (Author)
Department of Genetics,Stanford University

Use 20 ng/ml M-CSF instead of L-conditioned medium.

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9/2/2011 5:23:53 AM  

Alexandra Chung
University of Toronto

Hi,

Thank you for posting this protocol. I am starting to work with BM-derived macrophages and have followed other protocols, and I have been getting impure cultures where I see fibroblasts-like cells as well as round cells that I believe are monocytes/macrophages. In these cases, however, I did not culture with the L929 conditioned medium, but I used RPMI +FBS +M-CSF. I am just wondering if I follow this protocol, will I be able to get a pure culture of just macrophages, or is it impossible to do so?

Thank you for answering my question,
Alexandra

9/3/2011 2:53:42 AM  

Ran Chen (Author)
Department of Genetics,Stanford University

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.

8/30/2013 7:14:21 PM  

yu liming
Nanjing Medical University

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.

Reply

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