Bone Marrow Harvest from Mouse Hind Limb

Abstract

Source: Zhu, Y. P., Padgett, et al. Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells. J. Vis. Exp. (2019).

This video describes a detailed protocol to isolate fresh whole bone marrow from the mouse's hind limbs to investigate whole BM defects for patients with blood disorders such as leukemia.

Protocol

1. Harvest Mouse Bone Marrow (BM)

1. Purchase C57BL/6J mice from a commercial vendor. Feed the standard rodent chow diet and house in microisolator cages in a pathogen-free facility.
2. Use male mice, 6-10 weeks of age, for experimental purpose. Euthanize by CO₂ inhalation followed by the cervical dislocation.
3. Place the mouse onto a sterile surgical pad with the abdomen side up. Sterilize the skin of the abdomen and hindlimbs area by spraying 70% ethanol. Use a pair of dissecting surgical scissors to cut the abdominal cavity open.
4. Remove the skin to expose hindlimbs. Use a pair of blunt-tip dressing forceps to hold the mouse tibia right below the ankle. Use another pair of curved dressing forceps to stabilize the tibia below the blunt-tip dressing forceps. Break the tibia and expose the bone by ripping off the muscle with the blunt-tip dressing forceps.
   NOTE: The tibia is loosely attached to the knee joint and can be easily picked out by using the blunt-tip dressing forceps.
5. Place the tibia in cold 1x PBS.
6. Next, move the stabilizing curved dressing forceps downward to the femur. Slide the blunt-tip dressing forceps below the knee joint and hold the kneecap. Dislocate the kneecap by gently pulling it up. Expose the femur by ripping off the muscle attached to the kneecap. Hold the exposed femur by the curved dressing forceps and cut the femur off from the bottom of the bone using dissecting surgical scissors.
7. Place the femur in cold 1x PBS.
8. Punch a hole in 0.5 mL microcentrifuge tube with an 18 G needle.
9. Place both tibia and femur into the same 0.5 mL microcentrifuge tube with the open end of the bones facing downwards to the hole.
10. Place the 0.5 mL tube containing both tibia and femur into a 1.7 mL microcentrifuge tube.
11. Spin the double-layered tubes containing both tibia and femur at 5,510 x g for 30 s in the micro-centrifuge.
12. Ensure that the BM is extracted from the bones and pelleted at the bottom of the tube. Toss the 0.5 mL tube containing the hallowed bones. The mouse BM is ready for next steps.

Materials

Mouse: C57BL/6J	The Jackson Laboratory	Stock No: 000664
HyClone Phosphate Buffered Saline solution	GE Lifesciences	Cat#SH30256.01