Murine Hind Limb os long Dissection et moelle osseuse Isolation

The overall goal of this long bone dissection and bone marrow isolation procedure is to quickly and systematically remove the long bones and to collect the bone marrow for further downstream analysis. This method can help answer key questions related to bone marrow biology, cancer metastasis, and immunology. The main advantage of this technique is that the dissection process is quick and standardized, the bone marrow isolation procedure is fairly sterile, Begin by positioning the mouse in the supine position.

Then pin all four paw pads below the ankle joint and spray the mouse with 70%ethanol, thoroughly dousing the legs. Next, make a small incision to the right of the midline in the lower abdomen just above the hip, and extend the incision down the leg and past the ankle joint. Pull back the skin.

Then cut the quadriceps muscle anchored to the proximal end of the femur to expose the anterior side of the thighbone, and pin the muscle out from the leg with the pin at a 45 degree angle from the board. With the scissors against the posterior side of the femur, cut the hamstrings away from the knee joint. Then pull back the skin and hamstring muscles anchored to the proximal end of the femur to expose the posterior side of the bone, and pin the hamstring muscles out from the leg, placing the pin at a 45 degree angle.

With the forceps, grasp the distal end of the femur just above the knee joint. Then guide the scissor blades on either side of the femoral shaft toward the hip joint. After reaching the femoral head, twist the scissors, moving the top blade directly over the femoral head to dislocate the femur.

Now grasp the top of the femoral shaft with the forceps, and cut the soft tissue away from the femoral head to release the femur from the acetabulum. Then pull the entire leg bone, including the femur, knee, and tibia, up and away from the body, and carefully cut away the connective tissue and muscle attaching the leg to the skin. When all of the connective tissue has been removed, overextend the ankle joint and twist the scissors as just demonstrated to dislocate the tibia.

Grasping the distal end of the tibia and taking care not to sever the tendons, pull the tibia up and away from the body and the pin board. Then remove any remaining connective tissue attached to the long bone at the knee and any additional muscle or connective tissue attached to the femur and the tibia. To prepare the long bone for marrow isolation, grasp the femur with the patella facing away and the femoral head facing down.

Overextend the knee joint and twist the scissors to dislocate the tibia from the femur. Then remove any connective tissue holding the femur and tibia together. Next, grasp the femur with the anterior side facing away and the femoral head facing down, and guide the scissors up the femoral shaft to the condyles.

Gently rotate the scissors back and forth to remove the condyles, the patella, and the epiphysis, exposing the metaphysis. Then, use the forceps, scissors, and Kimwipes to remove any additional muscle or connective tissue attached to the femur. Next, grasp the tibia with the anterior side facing away and the ankle end facing down.

If the tibial epiphysis is intact, guide the scissors up the tibia shaft to the condyles and gently rotate the scissors back and forth to remove the condyles and epiphysis, exposing the tibial metaphysis. Then remove any additional muscle or connective tissue attached to the tibia, as just demonstrated. To harvest the bone marrow, first push an 18 gauge needle through the bottom of a 0.5 milliliter microcentrifuge tube.

Then place the long bones into the tube, knee end down, and close the lid. Nest the 0.5 milliliter microcentrifuge tube in a 1.5 milliliter microcentrifuge tube, and centrifuge the tubes at greater than or equal to 10, 000 times g in a microcentrifuge for 15 seconds. Verify that the bone marrow has been spun out of the bones by visual inspection.

The bones should appear white, and a large pellet should be visible in the larger tube. Then discard the bones in the 0.5 milliliter microcentrifuge tube, and suspend the bone marrow in the appropriate solution for the desired downstream analysis. This rapid dissection technique is suitable for a number of downstream analyses, including histomorphometry and histology.

Indeed, as demonstrated by this representative histomorphometric micro-CT 3D reconstruction, both the cancellous bone and the cortical shell are maintained, allowing an accurate quantitation of the standardized structural parameters for bone histomorphometry. In this representative histologic section of an H and E stained formal and fixed and decalcified tibia, the maintenance of the integrity of both the calcified bone and the cellular bone marrow for histologic analysis can be observed. Further, the bone marrow isolated by this procedure is suitable for many downstream applications, including the primary cell culture of osteoclasts or osteoblasts.

Once mastered, the technique can be completed in about seven minutes, from long bone dissection to the bone marrow isolation, if it is performed properly. While attempting the procedure, it's important to remember to position the mouse for the ease of dissection. Following this procedure, other methods, like FACS or other single cell processes can be performed to answer additional questions regarding alterations in the bone marrow cell populations.

After watching this video, you should have a good understanding of how to quickly dissect the long bones of mice and to isolate the bone marrow by centrifugation.