This kit contains recombinant Mouse M-CSF and Mouse RANKL, designed to induce the differentiation of mouse-derived precursor cells (such as the RAW264.7 cell line or primary bone marrow mononuclear cells) into mature osteoclasts with functional bone-resorbing capability.

M-CSF (Macrophage Colony-Stimulating Factor) supports the survival and proliferation of precursor cells and primes them for osteoclast differentiation, serving as the "licensing" signal. RANKL (Receptor Activator of Nuclear Factor Kappa-B Ligand) is the decisive signal driving the terminal differentiation and fusion of osteoclasts, activating key downstream transcription programs such as NFATc1.

The M-CSF and RANKL included in this kit are characterized by high activity, high purity, and low endotoxin levels. This kit efficiently drives the differentiation of precursor cells into osteoclasts, yielding a large number of mature, multinucleated, TRAP-strong positive osteoclasts.

Product Information

Components

Product Includes

Reconstitution Method

Storage

Long-term Storage: Store at -25 ~ -15°C. Valid for 1 year from the date of receipt.

Reconstituted (Short-term): After reconstitution, store under sterile conditions at 2 ~ 8°C for up to 7 days.

Reconstituted (Long-term): After reconstitution, store under sterile conditions at -85 ~ -65°C for up to 3 months.

Product Properties

Notes

1. For your safety and health, please wear a lab coat and disposable gloves during operation.

2. This product is for research use only.

Protocol

Protocol for Osteoclast Differentiation from RAW 264.7 Cells

1. Seeding of Osteoclast Precursors

1.1 Cell Preparation: Harvest RAW 264.7 cells in good growth condition and resuspend them in complete

α -MEM medium supplemented with 10% Fetal Bovine Serum (FBS). Perform cell counting.

1.2 Seeding: Seed the cells into a 24-well culture plate containing sterile glass coverslips at a density of

2.5×10³ cells/mL. Add 1 mL of cell suspension to each well.

1.3 Adherence: Incubate the plate in a humidified incubator at 37°C with 5% CO₂ for 18 hours to allow cell attachment.

2. Osteoclast Induction and Differentiation

2.1 Medium Replacement: Approximately 18 hours after seeding, once the cells have attached, carefully aspirate the old medium from the wells.

2.2 Induction: Replace with fresh complete osteoclast induction medium. This medium consists of α -MEM supplemented with 10% FBS, 50 ng/mL recombinant Mouse M-CSF Protein, and 100 ng/mL recombinant Mouse 2.3 RANKL Protein. Maintenance: Change the induction medium every 3 days. During each change, gently aspirate the spent medium and replace it with an equal volume of fresh induction medium containing the same concentrations of M-CSF and RANKL.

3. Morphological Observation and Identification

3.1 Morphology: Observe morphological changes regularly under an inverted microscope starting from the beginning of induction. Typically, typical multinucleated osteoclast-like structures can be observed after 4 days of RANKL induction.

3.2 TRAP Staining: After 5 days of RANKL induction, perform Tartrate-Resistant Acid Phosphatase (TRAP) staining to specifically identify osteoclasts. Strictly follow the manufacturer's instructions for the TRAP staining kit.

Result Interpretation: Observe under a light microscope after staining. Mature osteoclasts will exhibit a red or reddish-purple cytoplasm.

Documents:

Manuals:

92649_Manual_HB20260409