Tom Masi
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.
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