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Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMM’phi’)
小鼠骨髓来源的巨噬细胞(BMM’phi’)的分离培养   

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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(隔离), Culture(培养), Bone marrow-derived macrophages(骨髓来源的巨噬细胞)

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.

简介

骨髓来源的巨噬细胞是一种可以从哺乳动物骨髓中分离的白细胞。 在该方案中,描述了一种方法,其中从小鼠腿骨(股骨和胫骨)分离骨髓细胞,然后使用L929条件培养基在大约10天培养物中分化为骨髓来源的巨噬细胞。

关键字:隔离, 培养, 骨髓来源的巨噬细胞

材料和试剂

  1. L929细胞
  2. RPMI 1640培养基(RPMI)(Life Technologies,Invitrogen TM ,目录号:11875-093)
  3. 胎牛血清(FBS)(Atlanta Biologicals,目录号:S10350)
  4. 青霉素/链霉素(P/S)(Life Technologies,Invitrogen TM,目录号:15140-122)
  5. DPBS(Life Technologies,Invitrogen TM,目录号:14190-250)
  6. 75%乙醇
  7. 骨髓生长培养基(见配方)
  8. BMM'phi'生长培养基(参见食谱)

设备

  1. 细胞培养孵化器
  2. 0.22μm过滤器
  3. 27g针和1ml注射器
  4. 剪刀和镊子
  5. 15ml细胞培养皿

程序

  1. L细胞条件培养基的制备:在RPMI + 10%FBS中培养具有初始50%汇合的L929细胞5天,收集培养基并用0.22μm滤器过滤,分装并储存在-20℃。
    注意:使用前,在50°C下孵育30分钟。
  2. 准备骨髓生长培养基和BMM'phi'生长培养基(见配方)
  3. 小鼠骨髓细胞的分离。
    1. 牺牲鼠标,将鼠标浸泡在75%乙醇中
    2. 将皮肤夹在中间,并从身体下部清除皮肤。
    3. 用剪刀从腿上清除组织,并从身体上分离
    4. 清洁骨盆和股骨的剩余组织,并在膝关节分离。 小心不要打破骨头。 重要的是要确保所有的组织从骨骼中去除,因为与其相关的细胞可能污染骨髓制备物并可能过度生长巨噬细胞。
    5. 将骨头浸入75%乙醇中5分钟,然后将其浸入DPBS中5分钟,并将其留在RPMI + P/S中直到下一步。
    6. 切断骨头的每一端。
    7. 使用填充有骨髓生长培养基的27g针/1ml注射器; 用定向到15ml细胞培养皿中的培养基的射流从骨的两端排出骨髓。
    8. 每3天更换一次培养基。 当在培养中,一些细胞变得附着,而其中许多仍然在悬浮液中生长,因此在新的培养皿中自旋 - 下降和再培养这些细胞。
    9. 约10天后,几乎所有细胞都附着BMM'phi,然后BMM'phi'生长培养基用于进一步培养和测试。

食谱

  1. 骨髓生长培养基
    1. 加入5毫升P/S到500毫升瓶RPMI。 取出100毫升,并保存在一个干净的500毫升瓶子
    2. 加入20ml热灭活的胎牛血清和40ml L-细胞条件培养基,并混合均匀。
  2. BMM'phi'生长培养基
    RPMI + 10%FBS + 10%L-细胞条件培养基

致谢

这项工作由杭州高新区5050项目,杭州市海外退学基金资助,浙江省ZJ1000项目资助。 该方案是在美国加利福尼亚州斯坦福大学遗传学系的Cohen实验室开发的[Chen等人(未公开)]。

参考文献

  1. Gordon,S。(2003)。 巨噬细胞的替代活化。 Nat Rev Immunol 3( 1):23-35。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, R. (2012). Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMM’phi’). Bio-protocol 2(3): e68. DOI: 10.21769/BioProtoc.68.
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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.
3/3/2016 6:57:59 PM Reply
Ran Chen
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 ...

3/6/2016 11:28:01 PM


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
8/28/2015 8:37:55 AM Reply
Ran Chen
Stanford University

Usually one time use.

9/1/2015 6:04:29 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.
2/3/2015 7:37:19 PM Reply
Ran Chen
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.

3/3/2015 1:29:55 AM


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.
2/3/2015 7:32:47 PM Reply
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?
9/8/2014 5:55:32 PM Reply
Ran Chen
Stanford University

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

9/12/2014 10:52:35 PM


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?
10/4/2013 8:10:52 AM Reply
Ran Chen
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%.

10/5/2013 11:40:22 PM


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 3:10:23 AM Reply
Ran Chen
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.

9/10/2013 8:44:35 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.

7/2/2014 10:39:35 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 . what`s the purpose of incubating FBS at 50 °C for 30 min before using.
9/4/2013 3:40:56 AM Reply
Ran Chen
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.

9/6/2013 10:14:42 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.

9/7/2013 6:27:12 PM


Ran Chen
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 12:46:24 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.

9/9/2013 2:02:00 AM


What is the purpose of immersing bones in ethanol? wouldn't this fix the marrow?
2/12/2013 8:54:29 AM Reply
RAN CHEN
Stanford

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

2/14/2013 3:08:40 PM


Hi thank you for the protocol

I was wondering if i can activate the cells i would have isolated with a substance like lipopolysaccharide?
1/11/2013 12:41:35 AM Reply
RAN CHEN
Stanford

Yes, you can activate the cells with LPS.

1/14/2013 4:39:40 PM


Intelligence and simplicity - easy to unedrsatnd how you think.
11/16/2012 9:49:43 AM Reply
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.
4/11/2012 10:12:28 PM Reply
Ran Chen
Stanford University

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

4/14/2012 1:54:25 PM


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
9/2/2011 5:23:53 AM Reply
Ran Chen
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.

9/3/2011 2:53:42 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 . I am confused about that.

8/30/2013 7:14:21 PM