Bone Marrow Macrophages

Overview

Preparation of bone marraow macrophages from mice. Stolen from Virgin lab. (DW,DS,and ZZ; 5/15/07)

Materials

• BM20
• BM10
• PBS/EDTA

Procedure

Day 0

1. Dissect out mouse femurs and tibias (total of four bones per mouse).
2. Flush the bone marrow from each bone with 5-10 cc of BM20 using a syringe and a 26G needle. Combine the bone marrow from a single mouse into a 50-mL conical tube. Vortex briefly, and bring total volume to 50mL.
3. Plate cells into non-TC treated 10-cm petri dishes (Fisherbrand Cat #08-757-12), 10mL per Petri dish, 5 Petri dishes/mouse
4. Incubate at 37°C/5% CO2.

Day 4

1. Add 8-10 mL BM20/ petri dish. Be careful not to slosh the cells at this stage since 20mL BM20 per Petri dish will be quite full.

Day 7

1. Remove media, add 5 mL cold 0.02% EDTA in PBS (Sigma E8008). Incubate at 4°C for 10 min, then use a cell scraper to remove the cells from the plastic.
2. Transfer cells to a 50-mL conical, spin (1k rpm, 10 min), and resuspend in BM10 for counting.
3. Plate the cells in BM10 (generally1.5 x 105 cells/ well in a 24 well-plate). Allow cells to rest for 3 days.

Day10

1. Cells are now considered “rested” and ready for use.

Notes

1. Primary macrophages don’t come off TC-treated plastic.

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References

Relevant papers and books

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