This experiment highlights the therapeutic value of Anti-c-fms antibody in diseases of inflammatory bone disruption, such as rheumatoid arthritis and periodontitis. The process by which bone marrow is extracted and used to generate osteoclast precursors, yields large quantities of pure osteoclasts, which can be used in multiple downstream applications. This protocol provide two alternative methods of osteoclastogenesis, either using RANKL ligand or TNF alpha.
In addition to that, this method provides insight into the effect of Anti-c-fms antibody on osteoclastogenesis. Visual demonstration of this procedure familiarizes the researcher with the process of dissection and obtaining bone marrow cells in large amounts. To begin fill a plate that will serve as a collection container with approximately 50 milliliters of alpha MEM, depending on the size of the container, and place it on ice.
Use pins to pierce through the palms and feet of a euthanized mouse to fix it in supine position, and spray thoroughly with 70%ethanol. Use sterile surgical scissors and tweezers to make a skin incision at the level of the hip and extend it down to the feet, exposing the muscles. Locate and cut the tendon attaching the quadriceps muscle.
Peel away the muscle and cut it at the level of the hip. Then locate the tendon attaching the hamstring muscle to the bone. Peel away the muscles exposing the bone, and free the hamstring from its attachment, making sure not to cut into the bone.
Position the scissors between the head of the femur and the joint space. Cut into it freeing the femur, but keeping the bone intact, which will enable detachment of the bones without risk of exposing the bone marrow. Cutaway the muscles attached to the tibia in a similar fashion.
Then cut the tibia free from its attachment at the ankle joint, while keeping the bone intact to avoid contamination. Use the scissor blades and lab wipes to scrape any remaining soft tissue from the femur and tibia and place them in the prepared plate with alpha MEM placed on ice. The dissection should be carried out carefully to minimize contamination.
That is why thorough soft tissue removal and bone localization are crucial. Fill a 30 gauge needle syringe with alpha MEM. Disconnect the tibia and femur away from the knee joint, and cut the ends of the long bone from both sides using scissors.
Use tweezers to hold the bone from the shaft. Insert the needle into the marrow space to the bone and slowly flush the content of the marrow into a six centimeter culture dish and repeat for all bones. The bone will turn white, indicating the extraction of all bone marrow content.
Use a 40 micrometer nylon cell strainer to strain the bone marrow extract into a 50 milliliter conical centrifuge tube. Centrifuge the tube at 300 times G for five minutes. Discard the supernatant.
To wash, add five milliliters of alpha MEM to the pellet. Pipette the cells to detach them, centrifuge, and repeat the wash for a total of two times. After counting these isolated cells, as described in the manuscript, seed 10 million cells per 10 milliliters, in a 10 centimeter culture dish, and add M-CSF to the cells.
Incubate the culture at 37 degrees Celsius, 5%carbon dioxide for three days. Remove the medium after three days, and wash the cells twice vigorously with 10 milliliters of PBS to remove nonadherent cells. Add five milliliters of room temperature 0.02%trypsin-EDTA and PBS, and incubate at 37 degrees Celsius, 5%carbon dioxide for five minutes.
Thoroughly pipette the cells to detach them. Observe the culture under a microscope to make sure the cells have been detached and appear rounded and floating in media. When the cells have been detached, inactivate the reaction by adding five milliliters of the alpha MEM.
Collect the cells into a 50 milliliter conical tube and centrifuge at 300 times G for five minutes. After discarding the supernatant, wash with five milliliters of alpha MEM, and centrifuge again at 300 times G for five minutes. Re-suspend the pellet in 10 milliliters of alpha MEM.
Seed 1 million cells per 10 milliliters in a 10 centimeter culture dish, and add M-CSF. Incubate the culture at 37 degrees Celsius, 5%carbon dioxide for three days. Harvest the attached cells, which represent BMMs as osteoclast precursors after three days, as done previously during generation of BMMs.
Seed the BMMs at 50, 000 cells per 200 micro liters of alpha MEM in a 96 well plate per well. To each well, add desired stimuli. And then add M-CSF for a final concentration of 100 nanograms per milliliter.
Add Anti-c-fms antibody to each well. Incubate the plate at 37 degrees Celsius, 5%carbon dioxide for four days, while changing media every other day for four days. And then proceed with staining and assays, as described in the manuscript.
The osteoclast formation in M-CSF and RANKL treated culture, with Anti-c-fms antibody, was evaluated. With 101, 000 nanograms per milliliter Anti-c-fms antibody, a significant decrease in the number of osteoclasts was observed compared to control. Whereas, with one in 10 nanograms per milliliter Anti-c-fms antibody, there was no significant decrease.
Then, the osteoclast formation in M-CSF and TNF alpha treated culture, with Anti-c-fms antibody, was evaluated. With 10, 100, 1, 000 nanograms per milliliter Anti-c-fms antibody, a significant decrease in the number of osteoclasts was observed compared to control, with no significant decrease at one nanogram per milliliter. And the M-CSF culture with 1, 10, 100 nanograms per milliliter Anti-c-fms antibody, there was no significant decrease in the number of osteoclast precursors compared to control.
However, at 1, 000 nanograms per milliliter, there was a significant decrease in the number of osteoclast precursors, indicating that a concentration as high as 1, 000 nanograms per milliliter is needed to significantly inhibit proliferation of osteoclast precursors. Make sure to free the bonds while keeping the bone intact, which will minimize the chance of contamination.
