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Generation of Mouse Bone Marrow-Derived Macrophages (BM-MFs)
培养老鼠骨髓巨噬细胞   

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Abstract

Generating mouse macrophages from bone-marrow progenitor cells is a useful tool to study biological functions of mouse macrophages. Macrophages are one of the major populations of phagocytes and play many different roles during inflammatory process initiation and termination.

Keywords: Phagocytes(吞噬细胞), Macrophages(巨噬细胞), In vitro(体外), M-CSF(M-CSF)

Materials and Reagents

  1. M-CSF-transduced L929 cells
  2. HI FBS (EuroClone, catalog number: EC S0180L )
  3. L-Glutamine (EuroClone, catalog number: EC B3000D )
  4. Penicillin/streptomycin (EuroClone, catalog number: EC B3001D )
  5. IMDM (EuroClone, catalog number: EC B2072L )
  6. Beta-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  7. M-CSF-transduced L929 growth supernatant
  8. Phosphate buffered saline (PBS) (EuroClone, catalog number: ECM9605AX )
  9. BMMFs culture medium/ conditioned medium (see Recipes)

Equipment

  1. Centrifuges
  2. 70 μm-wide cut-off cell strainer
  3. Non-treated cell culture plates
  4. Incubator (37 °C and 5% CO2)
  5. Fluorescence activated cell sortor (FACS)

Procedure

  1. Flush mouse tibiae and femurs with ice-cold PBS through a 70 μm-wide cut-off cell strainer.
  2. Centrifuge 5 min at 450 x g. Resuspend pelleted cells in conditioned medium (supplemented with 30% of growth supernatant of M-CSF-transduced L929 cells).
  3. Seed 7 x 106 cells in 100 x 20 mm non-treated cell culture plates in 10 ml of conditioned medium.
  4. Incubate at 37 °C and 5% CO2.
  5. Upon reaching confluence (approximately 6 days) use the cells or split adhered cells and seed 5 x 106 cells in 100 x 20 mm in non-treated cell culture plates in 10 ml of conditioned medium.
  6. BMMFs are ready for experimental use when the percentage of CD11b+ cells is higher than 90% as measured by FACS analysis.

Recipes

  1. BMMFs culture medium recipe (conditioned medium)
    HI FBS - 10%
    L-Gln - 2mM
    Penicillin/streptomycin - 50 U/ml 
    Beta-mercaptoethanol - 50 μM
    M-CSF-transduced L929 growth supernatant - 30%
    IMDM - to volume

Acknowledgments

This protocol is described in our Nature paper (Zanoni et al., 2009). This work was supported by grants from the CARIPLO Foundation, the European Commission 6th Framework Program (MUGEN and DC-THERA contracts), the European Commission 7th Framework Program (TOLERAGE and ENCITE contracts), the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the and the Italian Ministry of Education and Research (COFIN).

References

  1. Zanoni, I., Ostuni, R., Capuano, G., Collini, M., Caccia, M., Ronchi, A. E., Rocchetti, M., Mingozzi, F., Foti, M., Chirico, G., Costa, B., Zaza, A., Ricciardi-Castagnoli, P. and Granucci, F. (2009). CD14 regulates the dendritic cell life cycle after LPS exposure through NFAT activation. Nature 460(7252): 264-268.

简介

从骨髓祖细胞产生小鼠巨噬细胞是研究小鼠巨噬细胞的生物学功能的有用工具。 巨噬细胞是吞噬细胞的主要种群之一,并在炎症过程开始和终止期间发挥许多不同的作用。

关键字:吞噬细胞, 巨噬细胞, 体外, M-CSF

材料和试剂

  1. M-CSF转导的L929细胞
  2. HI FBS(EuroClone,目录号:EC S0180L)
  3. L-谷氨酰胺(EuroClone,目录号:EC B3000D)
  4. 青霉素/链霉素(EuroClone,目录号:EC B3001D)
  5. IMDM(EuroClone,目录号:EC B2072L)
  6. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  7. M-CSF转导的L929生长上清液
  8. 磷酸盐缓冲盐水(PBS)(EuroClone,目录号:ECM9605AX)
  9. BMMFs培养基/条件培养基(见配方)

设备

  1. 离心机
  2. 70μm截止细胞过滤器
  3. 未处理的细胞培养板
  4. 培养箱(37℃和5%CO 2)
  5. 荧光激活细胞分选机(FACS)

程序

  1. 用冰冷的PBS冲洗小鼠胫骨和股骨通过一个70微米宽的截止细胞过滤器。
  2. 在450×g离心5分钟。 在条件培养基(补充有30%的M-CSF转导的L929细胞的生长上清液)中重悬沉淀的细胞。
  3. 在10ml条件培养基中,在100×20mm未处理的细胞培养板中种7×10 6个细胞。
  4. 在37℃和5%CO 2下孵育
  5. 在达到融合(约6天)时,使用细胞或分裂粘附的细胞,并在10ml未处理的细胞培养板中,在10ml条件培养基中,在100×20mm中接种5×10 6个细胞。 />
  6. 当通过FACS分析测量的CD11b +细胞的百分比高于90%时,BMMF准备用于实验使用。

食谱

  1. BMMFs培养基配方(条件培养基)
    HI FBS-10%
    L-Gln-2mM
    青霉素/链霉素 - 50U/ml<
    β-巯基乙醇 - 50μM
    M-CSF转导的L929生长上清液 - 30%
    IMDM - 到量为

致谢

该协议在我们的自然论文中描述(Zanoni等人,2009)。 这项工作得到了CARIPLO基金会,欧盟委员会第六框架计划(MUGEN和DC-THERA合同),欧盟委员会第七框架计划(TOLERAGE和ENCITE合同),意大利航空公司 和意大利教育研究部(COFIN)。

参考文献

  1. Zanoni,I.,Ostuni,R.,Capuano,G.,Collini,M.,Caccia,M.,Ronchi,AE,Rocchetti,M.,Mingozzi,F.,Foti,M.,Chirico,G.,Costa ,B.,Zaza,A.,Ricciardi-Castagnoli,P。和Granucci,F。(2009)。 CD14通过NFAT活化调节LPS暴露后的树突细胞生命周期。 自然 460(7252):264-268。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zanoni, I., Ostuni, R. and Granucci, F. (2012). Generation of Mouse Bone Marrow-Derived Macrophages (BM-MFs). Bio-protocol 2(12): e225. DOI: 10.21769/BioProtoc.225.
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I am currently culturing BMDM and following this protocol. I did the bone marrow isolation yesterday and seeded the cells on 100 mm non-treated culture dishes. After looking at the cells today, there just seems to be a many undifferentiated cells on the plate (they do not have any processes, just bright, round cells). Is 1 day too early to expect to see any differentiation in the cells? Or might there by something wrong with my media? Any help is appreciated on what the cells are expected to look like 1 day after extraction.

All the best,
Muktha
2/28/2012 8:05:12 PM Reply