After decapitation, sterilize the body of the anesthetized neonate mouse with 70%ethanol. Using forceps and fine tipped surgical scissors, make an incision between the abdomen and hind legs. Then remove the skin by pulling it toward the foot of the hind limbs.
Scrape the connective tissue and muscle attached to the bones using sharp edged forceps. Cut the tibia and femur with scissors. Use forceps to place the tibia and femur in a MEM culture media tube on ice.
Transfer the bones to a 40 micron strainer with a collection tube. Crush the bones with a three milliliter syringe plunger against the strainer to release the marrow. Centrifuge the cell suspension at 350 G for five minutes at four degrees Celsius.
Aspirate the supernatant and gently re-suspend the cells in 0.2%sodium chloride to make a hypotonic solution for the lysis of erythrocytes. Immediately add an equal volume of 1.6%sodium chloride to make the solution isotonic. Centrifuge the supernatant and remove the lysis solution.
Re-suspend the cells in complete DMEM and count the cells on an automated cell counter.
