Before you start
- Use non-tissue-culture-treated dishes so macrophages can be lifted easily.
Reagents and materials
Totals are for 2 mice (+10% extra). Per-mouse (2 femurs + 2 tibias) amounts are in the middle column.
| Reagent | Per mouse (2 femurs + 2 tibias) | Total |
|---|---|---|
| Cold PBS or medium for flushing | 10 mL | 10 mL |
| BMDM medium (DMEM + 10% FBS + pen/strep + 20 ng/mL M-CSF)four 10 cm dishes, 10 mL each | 40 mL | 40 mL |
| Extra BMDM medium on day 3 | 20 mL | 20 mL |
| PBS + 5 mM EDTA, cold (harvest) | 20 mL | 20 mL |
| 10 cm non-tissue-culture-treated Petri dishes | 4 each | 4 each |
Buffers and recipes
BMDM medium per 500 mL
| DMEM (high glucose) | 445 mL |
| Heat-inactivated FBS (10%) | 50 mL |
| Penicillin–streptomycin 100× (1×) | 5 mL |
| Recombinant mouse M-CSF (20 ng/mL) | 10 µg |
| or L929-conditioned medium | 10–30% instead of M-CSF |
L929-conditioned medium varies by batch; test each batch.
Equipment
- Dissection tools
- 25–27 G needle and 10 mL syringe
- 70 µm strainer
- Cell scraper
Procedure
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Collect bones
Remove femurs and tibias, clean off muscle and keep in cold PBS. Briefly dip in 70% ethanol, then rinse.
Flush the marrow
Cut the ends and flush the marrow with medium using a 25–27 G needle into a tube.
Make a suspension
Pipette up and down, pass through a 70 µm strainer and centrifuge at 300 × g for 5 min. Red-cell lysis is optional.
Plate
Resuspend in BMDM medium and plate into 10 cm dishes (10 mL each; marrow from one mouse into ~4 dishes).
Feed on day 3
Add 5 mL fresh BMDM medium to each dish without removing the old medium.
Harvest on day 6–7
Remove medium, add cold PBS + 5 mM EDTA for 5–10 min, then gently scrape and collect.
Check purity
Stain F4/80 and CD11b; expect more than 90% double-positive cells.
Critical points
- Avoid over-confluence; it alters activation state.
- Rest cells for several hours after re-plating before stimulation.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Few adherent cells | Check M-CSF activity or the L929 batch; check plating density. |
| Low purity | Extend culture to day 7; remove non-adherent cells at day 3. |
Safety
Standard practice for primary mouse cells.
Further reading
- Toda G et al. Preparation and culture of bone marrow-derived macrophages from mice for functional analysis. STAR Protocols (2021).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.