Perform the following in a biosafety cabinet to minimize contamination:

1. Dissect out both the femurs and tibias.

2. Clip the proximal and distal ends of the bones.

3. Fill a syringe with media (DMEM/F12 & 20% FBS) with an appropriate gauge needle.

4. Insert the needle into one end of the bone and blow the bone marrow out into a tube. Repeat for all bones.

5. Use a serological pipet to aspirate up and down to break up clumps.

6. Pass through a 70-100um cell strainer to remove clumps/bone chips.

7. Plate the single cell suspension in flask(s).

8. Incubate at 37 degrees, 5% CO2 for 48 hours

9. Remove media and wash with PBS to remove RBCs/non-attached cells.

10. Replace with fresh media and grow until 80-90% confluent. Change media every 3 days.