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

Published: August 31, 2023

Abstract

Source: Marahleh, A., et al. Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro. J. Vis. Exp. (2019)

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

  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.

Divulgaciones

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.
α-MEM Wako with L-Glutamine and phenol red
Fetal Bovine Serum Biowest s1820-500 Fetal Bovine Serum French Origin
Culture dish Corning 100 mm x 20 mm style dish
96-well plate Thermofisher Scientific Nun clon Delta surface

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Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro. J. Vis. Exp. (Pending Publication), e21613, doi: (2023).

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