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Macrophages are differentiated from circulating blood monocytes and act as tissue-resident professional phagocytes. Macrophages function in both innate and adaptive immune systems of vertebrate animals. The cytokine macrophage colony-stimulating factor (M-CSF) is essential for the proliferation and differentiation of monocytes. Here, we described a simple method to induce the differentiation of mouse bone marrow-derived myeloid precusor cells into macrophages in the presence of M-CSF.

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Mouse Macrophage Differentiation by Induction with Macrophage Colony-Stimulating Factor
通过用巨噬细胞集落刺激因子(M-CSF)诱导小鼠巨噬细胞分化

免疫学 > 免疫细胞分离 > 维持和分化
作者: Ying Liu
Ying LiuAffiliation: Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center of Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, USA
For correspondence: ivyliu0506@hotmail.com
Bio-protocol author page: a814
Keqiang Chen
Keqiang ChenAffiliation: Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center of Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, USA
Bio-protocol author page: a815
Chunyan Wang
Chunyan WangAffiliation: Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center of Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, USA
Bio-protocol author page: a816
Wanghua Gong
Wanghua GongAffiliation: Basic Research Program, SAIC-Frederick, Frederick, USA
Bio-protocol author page: a817
Teizo Yoshimura
Teizo YoshimuraAffiliation: Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center of Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, USA
Bio-protocol author page: a818
Ji Ming Wang
Ji Ming WangAffiliation: Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center of Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, USA
For correspondence: wangji@mail.nih.gov
Bio-protocol author page: a819
 and Mingyong Liu
Mingyong LiuAffiliation 1: Laboratory of Molecular Immunoregulation, Cancer Inflammation Program, Center of Cancer Research, National Cancer Institute-Frederick, NIH, Frederick, USA
Affiliation 2: Department of Spine Surgery, Daping Hospital, Third Military Medical University, Chongqing, China
For correspondence: liu_mingyong@yahoo.com.cn
Bio-protocol author page: a820
Vol 3, Iss 17, 9/5/2013, 3496 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.893

[Abstract] Macrophages are differentiated from circulating blood monocytes and act as tissue-resident professional phagocytes. Macrophages function in both innate and adaptive immune systems of vertebrate animals. The cytokine macrophage colony-stimulating factor (M-CSF) is essential for the proliferation and differentiation of monocytes. Here, we described a simple method to induce the differentiation of mouse bone marrow-derived myeloid precusor cells into macrophages in the presence of M-CSF.
Keywords: Monocytes(单核细胞), Differentiation(区别), Macrophages(巨噬细胞)

[Abstract]

Materials and Reagents

  1. Mice of interest
  2. 1x DPBS (Lonza, catalog number: 17-512F )
  3. ACK lysing buffer (Lonza, catalog number: 10-548E )
  4. Dulbecco’s Modified Eagle’s Medium with high glucose and without L-glutamine (DMEM) (Lonza, catalog number: 12-614F )
  5. Fetal Calf Serum (FCS) (Thermo Scientific, catalog number: SH30070.03 )
  6. Murine M-CSF (Peprotech, catalog number: 315-02 )
  7. 100x Penicillin/Streptomycin (Invitrogen, catalog number: 10378-016 )
  8. Complete DMEM medium (see Recipes)

Equipment

  1. 40 μm nylon strainer (BD Falcon, catalog number: 352340 )
  2. Needle (Gauge #25)
  3. Centrifuge

Procedure

  1. Mouse bone marrow (BM) cells are harvested from femurs by syringe and needle (Gauge #25) with 5 ml 1x DPBS. (Cut two ends of femurs and rinse out the cells by 1x DPBS)
  2. Centrifuge the cell suspension at 200 x g at room temperature (RT) for 5 min.
  3. Remove the supernatant and suspend the cell pellet with 2 ml ACK lysing buffer for 1 min to deplete red blood cells.
  4. The cell suspension is directly filtered through a 40 μm nylon strainer (observe the cell lysate under the microscope to determine whether red blood cells are depleted completely. If there are still too many red blood cells, add more ACK lysing buffer until there are very few red blood cells).
  5. Wash the strainer with 2 ml 1x DPBS and centrifuge the filtered cell suspension at 200 x g at RT for 5 min.
  6. The cell pellet is washed once again with 1x DPBS and re-suspended in 15 ml complete DMEM medium with 20 ng/ml murine M-CSF in a 100 mm Petri dish (one femur cells per dish) in an incubator (37 °C, 5% CO2).
  7. After 3 days, half of the medium is replaced with fresh complete DMEM medium.
  8. On Day 4, the cells containing more than 80% CD11b+/F4/80+ macrophages are ready for further characterization and functional experiments.

Recipes

  1. Complete DMEM medium (505 ml)
    100 ml FCS
    5 ml 100x Penicillin/Streptomycin
    400 ml DMEM

Acknowledgments

This protocol has been adapted from Liu et al. (2013).

References

  1. Liu, Y., Chen, K., Wang, C., Gong, W., Yoshimura, T., Liu, M. and Wang, J. M. (2013). Cell surface receptor FPR2 promotes antitumor host defense by limiting M2 polarization of macrophages. Cancer Res 73(2): 550-560.

材料和试剂

  1. 感兴趣的小鼠
  2. 1×DPBS(Lonza,目录号:17-512F)
  3. ACK裂解缓冲液(Lonza,目录号:10-548E)
  4. 具有高葡萄糖和不含L-谷氨酰胺(DMEM)(Lonza,目录号:12-614F)的Dulbecco改良Eagle培养基
  5. 胎牛血清(FCS)(Thermo Scientific,目录号:SH30070.03)
  6. 鼠M-CSF(Peprotech,目录号:315-02)
  7. 100x青霉素/链霉素(Invitrogen,目录号:10378-016)
  8. 完成DMEM培养基(参见配方)

设备

  1. 40μm尼龙过滤器(BD Falcon,目录号:352340)
  2. 针(量规#25)
  3. 离心机

程序

  1. 通过注射器和针(量规#25)用5ml 1x DPBS从股骨收获小鼠骨髓(BM)细胞。 (剪下股骨的两端并用1x DPBS冲洗细胞)
  2. 在室温(RT)下将细胞悬浮液以200×g离心5分钟。
  3. 除去上清液和悬浮细胞沉淀用2毫升ACK裂解缓冲液1分钟,以消耗红细胞。
  4. 细胞悬浮液通过40μm尼龙过滤器直接过滤(在显微镜下观察细胞裂解物以确定红细胞是否完全耗尽,如果仍然有太多的红细胞,则加入更多的ACK裂解缓冲液,直到非常少红细胞)。
  5. 用2ml 1×DPBS洗涤过滤器,并在室温下以200×g离心过滤的细胞悬浮液5分钟。
  6. 用1x DPBS再次洗涤细胞沉淀,并在培养箱(37℃,37℃)中在100mm培养皿(每皿中一个股骨细胞)中重悬于15ml含有20ng/ml鼠M-CSF的完全DMEM培养基中, 5%CO 2)。
  7. 3天后,将一半培养基用新鲜的完全DMEM培养基替换。
  8. 在第4天,含有超过80%CD11b + sup/+/fd/80 + 巨噬细胞的细胞准备用于进一步的表征和功能实验。

食谱

  1. 完全DMEM培养基(505ml)
    100 ml FCS
    5ml 100x青霉素/链霉素 400 ml DMEM

致谢

该协议已经从Liu等人改编(2013)。

参考文献

  1. Liu,Y.,Chen,K.,Wang,C.,Gong,W.,Yoshimura,T.,Liu,M.and Wang,J.M。 细胞表面受体FPR2通过限制巨噬细胞的M2极化来促进抗肿瘤宿主防御。 Cancer Res 73(2):550-560。
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How to cite this protocol: Liu, Y., Chen, K., Wang, C., Gong, W., Yoshimura, T., Wang, J. M. and Liu, M. (2013). Mouse Macrophage Differentiation by Induction with Macrophage Colony-Stimulating Factor . Bio-protocol 3(17): e893. DOI: 10.21769/BioProtoc.893; Full Text



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