Bone Marrow Harvest from Mouse Hind Limb

- Bone marrow is the spongy gelatinous tissue present in the hollow center of bones. It contains hematopoietic stem cells, which produce the blood cells, a key component of the immune system. Bone marrow isolation from larger bones, such as the femur and tibia, provides high cell yield.

To harvest bone marrow, place a euthanized mouse in a supine position and sterilize the skin using ethanol. Cut the abdominal cavity and remove the skin to expose the hind limbs. Break the tibia, a long bone of the leg, and place it in cold PBS to maintain the physiological conditions. Dislocate the kneecap and remove the muscles to expose the femur, the thigh bone.

Cut the femur off from the bottom of the bone and place in cold PBS. Punch a hole in a smaller volume microcentrifuge tube and place both bones in it with the bones' open-end facing the hole. Place the tube containing the bones into a larger volume microcentrifuge tube and centrifuge the double-layered tubes to pellet the bone marrow. Use the harvested bone marrow for further analysis. In this protocol, we will harvest bone marrow from the hind limbs of a mouse.

- For a bone marrow harvest, place a 6 to 10 week old C57 black-6 mouse in the supine position on a sterile surgical pad and use 70% ethanol to sterilize the abdomen and hind limbs. Use a pair of dissecting surgical scissors to open the abdominal cavity and remove the skin to expose the hind limbs. Using a pair of blunt tip dressing forceps, hold the mouse tibia right below the ankle and use a pair of curved dressing forceps to stabilize the tibia below the blunt tip dressing forceps.

Use the blunt tip dressing forceps to break the tibia and remove the muscle to expose the bone, before placing the tibia in cold PBS. Move the stabilizing curved dressing forceps to the femur and slide the blunt tip dressing forceps below the knee joint to hold the kneecap. Gently pull up to dislocate the kneecap and remove the muscle from the kneecap to expose the femur. Hold the exposed femur by the curved dressing forceps and use surgical scissors to cut the femur from the bottom of the bone. Then, place the femur in PBS.

Next, use an 18-gauge needle to punch a hole in a 0.5 milliliter microcentrifuge tube and place both bones in the tube with the open ends of the bones facing downward into the hole. Place the 0.5-milliliter tube into a 1.7 milliliter microcentrifuge tube and spin the double-layered tubes in a microcentrifuge. At the end of the centrifugation, confirm that the bone marrow has been extracted to the bottom of the tube.