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.


Method:
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 www.clodrosome.com 







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