Raw264.7 Osteoclast Differentiation Cytokine Kit

Osteoclast Precursor Cell Seeding
1.1 Resuspend RAW 264.7 cells in good growth condition in α-MEM complete medium supplemented with 10% fetal bovine serum (FBS), and perform a cell count.
1.2 Seed the cells at a density of 2.5 × 10³ cells/mL into a 24-well cell culture plate pre-loaded with sterile glass coverslips. Add 1 mL of the cell suspension to each well.
1.3 Incubate the plate in a 37°C, 5% CO₂ cell culture incubator for 18 hours to allow for complete cell adhesion.

Osteoclast Induction 
2.1 After cell adhesion (approximately 18 hours post-seeding), aspirate the old medium from the wells.
2.2 Replace with fresh osteoclast induction complete medium. The induction medium consists of α-MEM medium supplemented with 10% FBS, 50 ng/mL recombinant mouse M-CSF protein, and 100 ng/mL recombinant mouse RANK L protein.
2.3 Replace the induction medium every 3 days. During each change, gently aspirate the old medium and add an equal volume of fresh induction medium containing the same concentrations of M-CSF and RANK L proteins.

Cell Morphological Observation and Identification
3.1 Morphological Observation: Observe changes in cell morphology regularly under an inverted microscope starting from the initiation of induction. Typically, typical multinucleated osteoclast-like structures can be observed after 4 days of RANK L induction.
3.2 TRAP Staining Verification: After 5 days of RANK L induction, perform tartrate-resistant acid phosphatase (TRAP) staining to specifically identify osteoclasts. Strictly follow the instructions of the TRAP staining kit for the staining procedure. Upon completion of staining, observe under a light microscope. The cytoplasm of mature osteoclasts appears red or purple-red.