Bone Marrow Macrophages

Overview
Preparation of bone marraow macrophages from mice.

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 (generally 1.5 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.

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