Materials and Reagents

1. Cell scraper (Corning/VWR, catalog number: 15621-010 )

2. 70 µm nylon sterile cell strainer (Corning, catalog number: 352350 )

3. G18 needle (BD Bioscience, catalog number: 305196 )

4. 0.5 ml microfuge tube (Eppendorf, catalog number: 022363611 )

5. 1.5 ml microfuge tube (Eppendorf, catalog number: 022363204 )

6. 50 ml Falcon tube, screw top (Eppendorf, catalog number: 0030122178)

7. LC columns (Miltenyi, catalog number: 130-042-401 )

8. Non-tissue culture treated 6-well plates (Corning, catalog number: 351146 )

9. Serological pipettes (e.g., Fisher scientific catalog number: 10710810 )

10. Low adherent plates (Petri-dish) (VWR, catalog number: 25384-326 )

11. DMEM high glucose media (Gibco, catalog number: 11965092 )

12. (Optional) EasySep^TM (Stemcell Technologies, catalog number: 19861 )

13. Penicillin/Streptomycin (Gibco, catalog number: 15070063 )

14. Recombinant murine Macrophage Colony Stimulating Factor (rmM-CSF) (Genentech)

15. GlutaMAX (Gibco, catalog number: 35050061 )

16. Fetal Calf Serum (FCS) (Thermo Fisher, catalog number: 26140 )

17. Ammonium Chloride Potassium (ACK) lysis buffer (Thermo Fisher, catalog number: A1049201 )

18. Sterile Phosphate Buffered Saline (PBS) (Genentech)

19. Monocyte isolation kit (Miltenyi, catalog number: 130-100-629 )

20. Recombinant murine Granulocyte Macrophage Colony Stimulating Factor (rmGM-CSF) (R&D Systems, catalog number: 415-ML010 )

21. Recombinant murine Interleukin 4 (rmIL-4) (R&D Systems, catalog number: 404-ML-010 )

22. Macrophage media (see Recipes)

Equipment

1. Pipette boy (e.g., Integra, catalog number: 155017 )

2. Tabletop centrifuge (e.g., Beckman, model: Allegra X14 )

3. Microcentrifuge (e.g., Thermo Fisher Scientific, catalog number: 75002404 )

4. Water-bath at 37 °C (Thermo Fisher Scientific, catalog number: TSCIP19 )

5. Laminar Flow Hood (VWR)

6. Incubator at 37 °C with 5% CO₂ (e.g., Heracell 150 CO₂ incubator)

7. Vi-CELL XR Cell Viability Analyzer (Beckmann)

8. MACS Multi Stand magnet (Miltenyi, catalog number: 130-042-303 )

9. Sterile Scissors (Thermo Fisher Scientific, catalog number: 08-950 )

10. Sterile Forceps (Thermo Fisher Scientific, catalog number: 08-890 )

Procedure

1. Bone marrow isolation (Figure 2)

   1. Euthanize mouse using institutionally approved protocols.

   2. Use sterile forceps and scissors to isolate femur and tibia.

   3. Remove residual muscle or tissue attached to the bones.

   4. Store isolated bones in a 6-well plated in PBS on ice until further processing.

   5. Push a G 18 needle through the bottom of a 0.5 ml microcentrifuge tube to generate a small hole.

   6. Place the 0.5 ml microcentrifuge tube in a larger 1.5 ml microcentrifuge tube.

   7. Carefully place the isolated femur and tibia (maximum 1 femur and 2 tibia) in the 0.5 ml microcentrifuge tube nested in the 1.5 ml tube and close the lid.

   8. Centrifuge the tubes at 10,000 × g for 30 s.

   9. After centrifugation confirm complete transfer of marrow into the bottom tube. If transfer was not successful, cut off epiphysis and repeat centrifugation step.

   10. Discard the 0.5 ml microcentrifuge tube containing the bones. They should be empty of marrow.

   11. Resuspend the obtained bone marrow in 1 ml ACK lysis buffer and incubate at room temperature for 2 min.

   12. Place a 0.70 µm sterile filter on a 50 ml Falcon tube.

   13. Filter the bone marrow suspension into tube and wash the filter with 10 ml PBS.

   14. Add an additional volume of 30 ml PBS through the filter; discard the filter.

   15. Close the lid on the tube and centrifuge at 350 × g for 4 min.

   16. Remove PBS by gently decanting; retain bone marrow pellet.

   17. If desired, repeat PBS wash by gently resuspending the pellet.

   18. Count isolated bone marrow cells using a Vi-CELL or other standard method.

       
       

   
   Figure 2. Experimental outline depicting bone marrow isolation steps

   
   

2. Isolation and culture of monocytes

   1. Following bone marrow isolation, monocytes are enriched by negative selection using the monocyte isolation kit (Miltenyi) according to manufacturer’s instructions. Please note that other mouse monocyte isolation kits can be used for this step (e.g., EasySep^TM, Stemcell Technologies); however, these have not been evaluated for this specific protocol.

   2. Isolated monocytes can be cultured in macrophage media supplemented with 5 ng/ml rmGM-CSF and 2.5 ng/ml rmIL-4 for up to 5 days. Monocytes should be plated at a density of 1 × 10⁶ cells in 2 ml final volume in a non-tissue culture treated 6-well plate.

   3. If desired, monocytes can be enriched from the day 5 culture by sorting for Ly6C positive, F4/80 negative, MHCII negative and CD11c negative cells at day 5 (Figure 3).

      
      

   
   Figure 3. Flow cytometry analysis of freshly isolated and cultured monocytes. Example of surface marker expression of Ly6C, F4/80, CD11b and I-A/I-E of freshly isolated and cultured monocytes. Monocytes are identified as Ly6C ^high, F4/80 ^low, MHCII ^low and CD11c ^low.

   
   

3. Differentiation into bone marrow derived macrophages (BMDMs)

   1. Supplement macrophage media with 50 ng/ml rmM-CSF and pre-warm in a water bath to 37 °C.

   2. Suspend 12 million isolated total bone marrow cells (from Procedure A) or enriched monocytes (Procedure B) in 20 ml supplemented macrophage media and plate cells in 15 cm non-tissue culture treated plates.

   3. Place dish in cell culture incubator at 37 °C and 5 % CO₂ and culture for a total of 5 days.

   4. At day 3 add 20 ml freshly supplemented macrophage media without removal of the original media.

   5. At day 5 confirm adherence of differentiated BMDMs under a microscope. Assess differentiation by flow cytometry (Figure 4).

      
      

   
   Figure 4. Flow cytometry analysis of monocytes differentiated into bone marrow derived macrophages (day 5). Differentiated, resting macrophages can be identified as Ly6C ^low, F4/80 ^{high, CD11c low and I-A/I-E low.}

   
   

   6. For downstream assays, remove media and gently wash day 5 cells in 20 ml sterile PBS. Carefully scrape cells off the dish using a cell scraper in 5 ml macrophage media. If desired, wash the dish with an additional 5 ml of macrophage media to collect additional cells.

   7. Count collected cells and re-suspend in macrophage media supplemented with 50 ng/ml rmM-CSF at desired cell density (typical density is 0.5 x 10⁶ cells/ml). Plate cells onto tissue-culture treated dishes of desired formats.

   8. If desired, resting macrophages can be enriched from the day 5 culture by sorting for Ly6C ^low, F4/80 ^{high, CD11c low and I-A/I-E low.}

Recipes

1. Macrophage medium

   DMEM high glucose media

   10% FBS

   1× GlutaMAX

   1× Penicillin/Streptomycin

Acknowledgments

This study was funded by Genentech, Inc. References 8 and 9 have utilized the procedure for murine BMDM isolation and culture.

Competing interests

SMH and AM are employees of Genentech, Inc. AM is a shareholder in Roche.

Ethics

All mice and experiments in this study were conducted following protocols approved by Genentech Institutional Animal Care and Use Committee (Protocol ID TH19-0370; valid until 02/14/2022).

References

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3. Davies, J. Q. and Gordon, S. (2005). Isolation and culture of murine macrophages. Methods Mol Biol 290: 91-103.
4. Francke, A., Herold, J., Weinert, S., Strasser, R. H. and Braun-Dullaeus, R. C. (2011). Generation of mature murine monocytes from heterogeneous bone marrow and description of their properties.J Histochem Cytochem 59(9): 813-825.
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8. Freund, E. C., Lock, J. Y., Oh, J., Maculins, T., Delamarre, L., Bohlen, C. J., Haley, B. and Murthy, A. (2020). Efficient gene knockout in primary human and murine myeloid cells by non-viral delivery of CRISPR-Cas9.J Exp Med 217(7).
9. Lim, J., Park, H., Heisler, J., Maculins, T., Roose-Girma, M., Xu, M., Mckenzie, B., van Lookeren Campagne, M., Newton, K., and Murthy, A. (2019). Autophagy regulates inflammatory programmed cell death via turnover of RHIM-domain proteins. Elife 8: e44452.