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

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 ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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