Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

1. Generation of BMM

1. Seed 1 x 10⁷ cells/10 mL in a 10 cm culture dish and add M-CSF 100 ng/mL. The final concentration of M-CSF is 100 ng/mL of culture medium. Incubate the culture at 37 °C, 5% CO₂ for 3 days.
2. After 3 days, remove the culture medium, wash the cells vigorously with 10 mL of phosphate-buffered saline (PBS) twice to remove non-adherent cells, add 5 mL of room temperature 0.02% trypsin-EDTA in PBS, and incubate at 37 °C, 5% CO₂ for 5 min.
3. Detach the cells by thorough pipetting. Make sure that the cells have been detached by observing the culture under a microscope (the cells should appear rounded and floating in media). If the cells remain attached, repeat pipetting thoroughly to detach them. When the cells have been detached, add 5 mL of α-MEM to inactivate the reaction.
4. Collect the cells into a 50 mL conical tube and centrifuge at 300 x g for 5 min. Discard the supernatant, and wash with 5 mL of α-MEM.
5. Centrifuge at 300 x g for 5 min and resuspend the pellet in 10 mL of α-MEM.
6. Perform a cell count.
7. Seed 1 x 10⁶ cells/10 mL in a 10 cm culture dish and add M-CSF 100 ng/mL. The final concentration of M-CSF is 100 ng/mL of culture medium. Incubate the culture at 37 °C, 5% CO₂ for 3 days.

2. Generation of Osteoclasts from BMM as Osteoclast Precursors

1. After 3 days, harvest the attached cells which represent BMM as osteoclast precursors, following steps 1.2-1.7.
2. Seed BMM at 5 x 10⁴ cells/200 μL of α-MEM in a 96-well plate. Add RANKL for a final concentration of 50 ng/mL or TNF-α for a final concentration of 100 ng/mL. Add M-CSF for a final concentration of 100 ng/mL to each well.
3. Add anti-c-fms antibody (final concentrations of 0, 1, 10, 100, 1000 ng/mL) to each well.
4. Incubate at 37 °C, 5% CO₂ for 4 days. Change media every other day for 4 days.

3. Tartrate-resistant Acid Phosphatase (TRAP) Stain

1. After 4 days of incubation, gently aspirate the culture medium of each well. Wash each well once with 200 μL of room temperature 1x PBS.
2. Add 200 μL of 10% formalin to each well for fixation and incubate for 1 h at room temperature. Aspirate the formalin and wash each well 3 times with deionized water.
3. Add 200 μL of 0.2% Triton X-100 in PBS to each well for permeabilization and incubate for 1 h at room temperature. Aspirate the Triton X-100 and wash each well 3 times with deionized water.
4. Add 200 μL of TRAP stain solution to each well. Visualize the stain under the microscope. It will take from 10-30 min for the stain to develop properly.
5. Aspirate the stain and wash each well with deionized water 3 times, and air dry. Keep it at room temperature to use in further observation.