Original research article

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Jul 2009

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Generation of Mouse Bone Marrow-Derived Macrophages (BM-MFs)    

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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, Macrophages, In vitro, 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)


  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)


  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.


  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


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).


  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.
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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,
2/28/2012 8:05:12 PM Reply
songzi liu
WuHan University

Our lab seed cells in 12-well plates at 1 *10^6 cells per wall concentration. Usually, we change culture medium every 2 days and differentiation may last 7 days.

11/19/2018 4:43:45 PM

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