Cytokine Kit for Inducing Osteoclast Differentiation of Mouse Bone Marrow Cells (BMMs)

1ml system：M-CSF need 180ng in all , RANKL total require 150ng

1.Bone Marrow Cell Seeding

1.1After red blood cell lysis of freshly isolated mouse bone marrow cells, resuspend the cells in α-MEM complete medium containing 10% fetal bovine serum (FBS) and perform cell counting.

1.2Dilute the cell density to 2 × 10⁵ cells/mL, add M-CSF to a final concentration of 30 ng/mL, and seed the cells into 24-well cell culture plates pre-loaded with sterile coverslips, with 1 mL of cell suspension added to each well.

1.3 Incubate the plates statically in a cell incubator at 37°C with 5% CO₂ for 5 days, and change the medium (containing 30 ng/mL M-CSF) every 2 days.

2.Osteoclast Induction and Differentiation

2.1Aspirate and discard the old medium in the wells. Add complete medium containing 30 ng/mL M-CSF to the negative wells, and add complete medium containing 30 ng/mL M-CSF and 50 ng/mL RANKL protein to the positive wells.

2.2Thereafter, change the medium every 2 days. Add complete medium containing 30 ng/mL M-CSF to the negative wells, and add complete medium containing 30 ng/mL M-CSF and 50 ng/mL RANKL protein to the positive wells.

3.Morphological Observation and Identification of Cells

3.1 Morphological observation: Regularly observe the changes in cell morphology under an inverted microscope starting from the induction. Typically, osteoclast-like structures can be observed 4 days after RANKL induction.

3.2 TRAP staining verification: Perform tartrate-resistant acid phosphatase (TRAP) staining to specifically identify osteoclasts 5–7 days after RANKL induction. Conduct the staining strictly in accordance with the instructions of the TRAP staining kit.

Observe the cells under a light microscope after staining. The cytoplasm of mature osteoclasts appears red or reddish-purple.