Wednesday, January 11, 2012

Isolation of osteoclasts from murine bone

Osleoclasts are macrophage-like cells that are located in the bones. Osteoclasts are specialized for bone resorption. Due to the frequent cells fusion, osteoclasts are multinucleated.  Osteoclasts stain positively for tartrate-resistant acid phosphatase (TRAP)  after 3 days in culture. Osteolclasts exist in low number in bones. The protocol below describes the enrichment of these cells.

1) Sacrifice the mice and bleed them by aortic section.
2) Dissect mouse femurs and tibias from 7-10 day old mice.
3) Remove all the soft tissues from the bones.
4) split the bone longitudinally.
5) Remove and discard the bone marrow and grind the bones using a small grinder. 
6) Suspend the bone fragments in 1 ml of supplemented medium 199 (Invitrogen) (mebium 199, 15% Fetal Calf Serum, 100 U/ml penicillin, 100 ug/ml streptomycin) 
7) Pour 0.2 ml aliquots into 6-well tissue culture plate (Falcon).
8) Incubate the bone fragments at 37 degree centigrade, 5% carbon dioxide for 1 hour.
9) Remove the non-adherent cells and bone fragments by rinsing the plates with PBS (Sigma)
10) Add 5 ml of fresh supplemented medium 199.

For more information about macrophage depletion visit 

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