In this video, we demonstrate the effect of different anti-c-fms antibody concentrations on osteoclastogenesis, which is the process of osteoclast formation from osteoclast progenitors.
Protocol
1. Generation of BMM
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.
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.
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.
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.
Centrifuge at 300 x g for 5 min and resuspend the pellet in 10 mL of α-MEM.
Perform a cell count.
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
After 3 days, harvest the attached cells which represent BMM as osteoclast precursors, following steps 1.2-1.7.
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.
Add anti-c-fms antibody (final concentrations of 0, 1, 10, 100, 1000 ng/mL) to each well.
Incubate at 37 °C, 5% CO2 for 4 days. Change media every other day for 4 days.
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.
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.
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.
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.
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.
Declarações
The authors have nothing to disclose.
Materials
Anti-c-Fms antibody
AFS98, a rat monoclonal, antimurine, c-Fms antibody (IgG2a)
RANKL
PEPROTECH
315-11
Recombinant Murine sRANK Ligand, Source: E.coli
M-CSF
Recombinant human M-CSF. 1/10 vol of CMG14–12 cell line culture supernatant at 5X106 cells in a 10-cm suspension culture dish.