Recombinant Mouse Fractalkine (FKN)

Recombinant Mouse Fractalkine (FKN, CX3CL1) is a valuable tool for research applications focused on immune cell migration, neuroinflammation, tissue regeneration, and disease modeling, due to its unique dual role as both a chemoattractant and cell adhesion molecule for CX3CR1-expressing cells.

Key scientific applications and rationale:

• Immune Cell Migration and Adhesion: FKN specifically binds to the CX3CR1 receptor, regulating the migration and adhesion of monocytes, T cells, and NK cells. This makes recombinant FKN essential for studying leukocyte trafficking, inflammatory responses, and immune surveillance in vitro and in vivo.

• Neuroinflammation and Neuroprotection: Recombinant FKN has been shown to attenuate neuroinflammation, promote hematoma resolution, and improve neurological outcomes in mouse models of brain injury by modulating microglial polarization toward an anti-inflammatory (M2) phenotype. It also supports neuron–glia interactions and can be used to investigate mechanisms of neurodegeneration and neuroprotection.

• Remyelination and Regeneration: In mouse models of multiple sclerosis, FKN administration promotes remyelination by stimulating oligodendrocyte precursor cell differentiation and reducing pro-inflammatory immune cell infiltration, suggesting its utility in studies of CNS repair and regeneration.

• Transplantation and Ischemia-Reperfusion Injury: The FKN–CX3CR1 axis plays a major role in graft rejection and ischemia-reperfusion injury. Recombinant FKN can be used to model these processes, test interventions, and study mechanisms underlying immune-mediated tissue damage.

• Chemoattractant Bioassays: Recombinant FKN is used in chemotaxis assays to quantify the migration of CX3CR1+ cells, with established ED50 values for attracting human lymphocytes and mouse pro-B cells.

• Metabolic and Inflammatory Disease Models: Chronic administration of FKN improves glucose tolerance and modulates inflammatory responses, making it relevant for metabolic disease and rheumatic disease research.

Best practices for use:

• In vitro: Employ recombinant FKN in cell migration, adhesion, and signaling assays to dissect CX3CR1-dependent pathways.
• In vivo: Use recombinant FKN for direct administration in mouse models to study its effects on immune cell dynamics, neuroinflammation, tissue regeneration, and disease progression.
• ELISA and Standards: Recombinant FKN serves as a standard for quantifying endogenous fractalkine levels in biological samples.

Summary of scientific value:
Using recombinant mouse fractalkine enables precise, reproducible investigation of CX3CR1-mediated processes in immunology, neuroscience, and regenerative medicine, facilitating mechanistic studies and therapeutic development.

Yes, recombinant Mouse Fractalkine (FKN, CX3CL1) can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and its concentration is accurately determined. This approach is standard practice in quantitative ELISA protocols for mouse Fractalkine.

Essential context and supporting details:

• ELISA Standard Practice: Most commercial mouse Fractalkine ELISA kits use recombinant mouse Fractalkine as the standard for generating calibration curves. The recombinant protein is serially diluted to create a standard curve, which is then used to quantify unknown samples by comparison.

• Purity and Source: The recombinant protein should be highly purified and free of contaminants that could interfere with antibody binding or detection. It is commonly produced in insect cells (e.g., Sf21) or other expression systems.

• Concentration Determination: Accurate quantification requires precise measurement of the recombinant protein’s concentration, typically using spectrophotometric methods or amino acid analysis. The standard curve should cover the expected range of sample concentrations (e.g., 0.3–200 ng/mL or 312–20,000 pg/mL, depending on kit sensitivity).

• Compatibility: The recombinant standard must be recognized by the antibodies used in your ELISA. Commercial kits are validated to ensure that both natural and recombinant mouse Fractalkine are detected equivalently. If you are developing your own assay, confirm antibody compatibility with your recombinant standard.

• Matrix Effects: When preparing standards, dilute the recombinant protein in the same buffer or matrix as your samples to minimize matrix effects and ensure accurate quantification.

• Regulatory Note: Recombinant standards and ELISA kits for mouse Fractalkine are intended for research use only, not for diagnostic or clinical procedures.

Additional relevant information:

• Validation: It is good practice to validate the linearity and recovery of your recombinant standard in your specific assay setup, especially if you are not using a commercial kit.

• Bioactivity: If you require the standard for bioactivity assays (e.g., cell migration), ensure the recombinant protein is suitable for such applications, as some preparations are intended only for ELISA calibration and not for functional assays.

• Storage: Follow recommended storage conditions for recombinant proteins to maintain stability and activity (typically at −20°C or 4°C, avoiding repeated freeze-thaw cycles).

In summary, recombinant Mouse Fractalkine is widely used and appropriate as a standard for ELISA quantification, provided it is properly validated and matched to your assay system.

Recombinant Mouse Fractalkine (FKN, CX3CL1) has been validated for several key applications in published research, primarily in functional bioassays, in vivo disease models, and mechanistic studies of immune and neuroinflammatory processes.

Validated Applications:

• Bioassays (Chemotaxis):

  • Recombinant mouse FKN is widely used to assess its ability to chemoattract cells expressing the CX3CR1 receptor, such as peripheral blood lymphocytes and transfected cell lines. The effective dose (ED50) for chemoattraction is typically reported in the range of 0.03–0.6 μg/mL, depending on the cell type and assay conditions.

• In Vivo Disease Models:

  • Neuroinflammation and Hemorrhage: Intranasal or systemic administration of recombinant FKN has been shown to promote hematoma resolution, attenuate neuroinflammation, and improve neurological outcomes in mouse and rat models of germinal matrix hemorrhage (GMH). These effects are mediated via the CX3CR1/AMPK/PPARγ signaling pathway and involve modulation of microglial polarization (M1/M2 phenotypes).
  • Transplantation and Ischemia-Reperfusion Injury: FKN has been used to study the role of the FKN-CX3CR1 axis in acute rejection and ischemia-reperfusion injury in mouse heart transplantation models. Recombinant FKN and anti-FKN antibodies have been applied to modulate monocyte migration and improve graft survival.
  • Osteoclast Differentiation: FKN regulates osteoclast precursor (OCP) survival and primes these cells for subsequent osteoclast differentiation in mouse models, indicating its use in bone biology and osteoimmunology research.

• Mechanistic Studies:

  • Microglia and Macrophage Polarization: Recombinant FKN is used to investigate its effects on microglial and macrophage polarization, neurotoxicity, and migration in models of neurodegeneration and CNS autoimmune inflammation.
  • Contact Hypersensitivity: FKN has been implicated in modulating macrophage polarization toward the M2 phenotype in models of contact hypersensitivity.

• Immunoassays and ELISA Standards:

  • Recombinant FKN serves as a standard antigen in ELISA assays for quantifying endogenous fractalkine levels in biological samples.

Summary Table:

Additional Notes:

• Recombinant mouse FKN is typically used in both in vitro and in vivo settings, with validated activity confirmed by chemotaxis and functional assays.
• Its role in modulating immune cell behavior makes it a valuable tool for dissecting mechanisms of inflammation, tissue injury, and repair.

If you require protocols or specific experimental details for any of these applications, please specify the context or model system.

To reconstitute and prepare Recombinant Mouse Fractalkine (FKN, CX3CL1) protein for cell culture experiments, first centrifuge the vial briefly to collect the lyophilized powder at the bottom. Then, reconstitute the protein at a concentration of 0.1–0.5 mg/mL in sterile distilled water or sterile PBS, depending on the formulation and manufacturer’s instructions. Avoid vigorous mixing or vortexing to prevent protein denaturation.

Step-by-step protocol:

• Centrifuge the vial before opening to ensure all lyophilized material is at the bottom.
• Reconstitute:
  • If the protein is lyophilized from PBS, use sterile PBS for reconstitution.
  • If the protein is lyophilized without buffer, use sterile distilled water.
  • For carrier-free formulations, reconstitute at 100 μg/mL in sterile PBS.
  • For formulations with BSA as a carrier, reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% BSA.
  • For general use, a concentration of 0.1–0.5 mg/mL is typical.
• Mix gently by pipetting up and down; do not vortex.
• Allow the protein to dissolve for 10–30 minutes at room temperature, gently swirling if needed.
• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
• Storage:
  • Store aliquots at –20°C to –70°C for long-term use.
  • After reconstitution, the protein is stable for 2–7 days at 2–8°C.
  • For long-term storage, adding a carrier protein (e.g., 0.1% BSA) is recommended to prevent adsorption and loss of activity.

Additional notes for cell culture:

• Use carrier-free protein if BSA or other additives may interfere with your assay.
• Filter-sterilize the final solution if sterility is required and not already ensured by the manufacturer.
• Prepare working dilutions in cell culture medium immediately before use.

Summary Table:

Always consult the specific product datasheet for any unique requirements or buffer components. If the protein is supplied in a buffer containing acetonitrile or TFA, ensure complete removal or dilution before adding to cells.