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 Product Information. Catalog Number Size Mouse Osteoclast Cell Differentiation Cytokine Set 92649ES10 10 μg 92649ES50 50 μg Components Component Cat No. Component Name 92649ES10 92649ES50 92649ES-A Mouse M-CSF Protein 10 μg 50 μg 92649ES-B Mouse RANKL Protein 25 μg 100 μg Product Includes Component Cat No. Product Name Recommended Concentration Source Appearance Quantity 91114ES Mouse M-CSF Protein 50 ng/mL E.coli Lyophilized Powder 1 95625ES Mouse RANKL Protein 100 ng/mL HEK293 Lyophilized Powder 1 Reconstitution Method Component Name Reconstitution Method Mouse M-CSF Protein Prior to opening the vial, centrifuge to ensure the contents are collected at the bottom. Reconstitute with sterile distilled water or PBS to a concentration of 0.1–1.0 mg/mL. The solution may be further diluted according to subsequent experimental requirements. For long-term storage, it is recommended to add a carrier protein (0.1% BSA) to the dilution buffer. Aliquot into single-use volumes and store at -80°C. Avoid repeated freeze-thaw cycles. Mouse RANKL Protein Centrifuge before opening. Reconstitute the lyophilized protein with distilled water to a concentration greater than 100 μg/mL. It is recommended to aliquot the protein into single-use portions upon first use to avoid repeated freeze-thaw cycles. 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 Source Species Mouse Tag No Tag Expression System HEK293 Endotoxin < 1.0 EU per μg by the LAL method. Formulation Lyophilized from 0.22 μm filtered solution in PBS (pH 7.4). Appearance Lyophilized Powder 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
