Recombinant Human CX3CL1/Fractalkine (FKN)

Recombinant human CX3CL1/fractalkine (FKN) offers several compelling advantages for research applications across multiple biological systems and disease models.

Dual Functional Capabilities

Recombinant FKN functions uniquely as both a chemokine and adhesion molecule. Unlike conventional chemokines, it possesses a distinctive structural architecture comprising a CX3C chemokine domain and a mucin stalk. This dual functionality allows the protein to mediate leukocyte binding and adhesion while simultaneously serving as a potent chemoattractant. The soluble chemokine domain can be shed from the mucin stalk to act independently, or remain membrane-bound, providing experimental flexibility depending on your research design.

Broad Immunological Applications

The CX3CL1-CX3CR1 axis plays a critical role in immune cell recruitment and activation. Recombinant FKN effectively attracts T cells, monocytes, and natural killer cells, making it valuable for studies investigating immune cell migration, infiltration, and inflammatory responses. This makes it particularly useful for research examining leukocyte behavior in inflammatory disease models.

Neuroprotective and Anti-inflammatory Properties

Recombinant FKN demonstrates significant neuroprotective effects in neurological applications. In germinal matrix hemorrhage models, recombinant FKN treatment promoted hematoma resolution, attenuated neuroinflammation, and improved neurological outcomes through the AMPK/PPARγ signaling pathway. The protein reduced pro-inflammatory cytokine release (IL-6, IL-1β, TNFα) while increasing anti-inflammatory cytokines (IL-10). Additionally, FKN reduced N-methyl-D-aspartate-induced calcium flux and apoptosis in human neurons through extracellular signal-regulated kinase activation.

Therapeutic Pathway Modulation

Recombinant FKN activates specific intracellular signaling cascades that can be precisely monitored in research settings. CX3CR1 activation by recombinant FKN promotes microglial polarization from pro-inflammatory M1 phenotypes toward anti-inflammatory M2 phenotypes, enabling detailed mechanistic studies of immune cell differentiation and function.

Disease Model Versatility

The CX3CL1-CX3CR1 axis has demonstrated relevance across diverse pathological conditions, including chronic kidney disease, diabetic nephropathy, renal fibrosis, atherosclerosis, traumatic brain injury, and systemic sclerosis. This broad applicability makes recombinant FKN a valuable tool for investigating common inflammatory and degenerative disease mechanisms.

Experimental Optimization

Recombinant FKN allows for dose-dependent studies, as moderate concentrations have been shown to produce optimal biological effects compared to both lower and higher doses. This enables researchers to establish dose-response relationships and identify optimal concentrations for specific experimental questions.

Yes, recombinant human CX3CL1/Fractalkine (FKN) 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 widely accepted in quantitative immunoassays for chemokines such as Fractalkine.

Supporting details:

• ELISA Calibration: Commercial ELISA kits for human Fractalkine routinely use highly purified recombinant human CX3CL1/Fractalkine as the calibrator standard. These standards are typically NS0-expressed, full-length recombinant proteins, and are validated to generate accurate, linear standard curves for quantification.
• Parallelism: Studies have shown that standard curves generated with recombinant Fractalkine are parallel to those obtained with natural Fractalkine, indicating that recombinant protein is suitable for determining relative mass values of endogenous Fractalkine in biological samples.
• Sample Types: Recombinant Fractalkine standards are used for quantification in diverse sample matrices, including serum, plasma, urine, cell culture supernatants, and saliva.
• Preparation: For use as a standard, the recombinant protein should be reconstituted and diluted in the same calibrator diluent as used for the assay, following the recommended protocol to ensure consistency and accuracy.
• Validation: It is important to confirm that the recombinant standard is recognized by the antibodies in your ELISA system and that it produces a linear, reproducible standard curve within the assay’s dynamic range.

Best practices:

• Use a recombinant standard that matches the form of Fractalkine detected by your assay (e.g., full-length or chemokine domain).
• Verify the protein’s purity and concentration using orthogonal methods (e.g., BCA assay, SDS-PAGE).
• Prepare a serial dilution series in the recommended calibrator diluent to generate the standard curve.
• Validate parallelism and recovery in your specific sample matrix if possible.

Limitations:

• Any variation in diluent, operator technique, incubation time, or temperature can affect quantification accuracy.
• The recombinant standard should be free of carrier proteins or additives that may interfere with antibody binding or detection.

Summary: Recombinant human CX3CL1/Fractalkine is scientifically appropriate and commonly used as a standard for ELISA quantification, provided it is properly validated and prepared according to assay requirements.

Recombinant Human CX3CL1/Fractalkine (FKN) has been validated in published research for a range of applications, primarily involving its roles in chemotaxis, cell adhesion, immune modulation, and disease modeling.

Key validated applications include:

• Bioassays for chemotaxis and adhesion: Recombinant CX3CL1 is widely used to study the chemotactic migration and adhesion of CX3CR1-expressing cells, such as T cells, monocytes, NK cells, and microglia, in both static and flow-based assays. These assays help elucidate the mechanisms of leukocyte recruitment and trafficking in inflammation and immunity.

• Functional studies in inflammation and immune response: CX3CL1 is used to model and dissect its role in inflammatory diseases (e.g., atherosclerosis, rheumatoid arthritis, glomerulonephritis), where it mediates leukocyte recruitment, adhesion, and activation. It is also used to test the effects of CX3CL1 blockade or supplementation in animal models of these diseases.

• Neuroscience research: Recombinant CX3CL1 is applied in studies of neuroinflammation and neurodegeneration, including models of Parkinson’s disease, traumatic brain injury, and spinal cord injury, to investigate its neuroprotective or neurotoxic effects on neurons and microglia.

• Cancer research: The protein is used to assess its dual roles in tumor biology, including recruitment of immune effector cells (antitumor immunity) and promotion of tumor cell adhesion, migration, and metastasis. It is also used in in vivo models to evaluate its impact on tumor growth and immune cell infiltration.

• Metabolic and endocrine studies: Recombinant CX3CL1 has been validated for protecting pancreatic β-cells from cytokine-induced damage and for modulating insulin secretion in studies of diabetes and metabolic syndrome.

• Osteoclast differentiation assays: It is used to study the differentiation of monocytes and dendritic cells into osteoclasts, relevant for bone biology and diseases such as osteoporosis.

• ELISA and immunoassays: While not a direct application of the recombinant protein itself, CX3CL1 is used as a standard or control in ELISA kits for quantifying endogenous fractalkine in biological samples.

Summary Table of Validated Applications

Experimental formats include in vitro cell-based assays, ex vivo tissue studies, and in vivo animal models, depending on the research question.

If you need protocols or more specific details for a particular application, please specify the context or experimental system.

To reconstitute and prepare Recombinant Human CX3CL1/Fractalkine (FKN) protein for cell culture experiments, follow these best practices based on standard protocols and manufacturer recommendations:

1. Preparation Before Reconstitution

• Briefly centrifuge the lyophilized protein vial at low speed (e.g., 1,000–2,000 × g for 1–2 minutes) to ensure all powder is at the bottom of the vial before opening.

2. Reconstitution

• Reconstitution Buffer: Use sterile, endotoxin-free PBS or sterile distilled water. If the protein is supplied with a carrier protein (e.g., BSA), reconstitute in PBS containing at least 0.1% BSA. For carrier-free (CF) versions, use sterile PBS only.
• Concentration: Reconstitute to a concentration of 25–100 µg/mL (or as specified by the manufacturer). For example:
  • For a carrier-containing version: Reconstitute at 25 µg/mL in sterile PBS with 0.1% BSA.
  • For carrier-free (CF) version: Reconstitute at 100 µg/mL in sterile PBS.
• Method: Gently add the appropriate volume of buffer to the vial. Avoid vigorous mixing; instead, swirl or let the vial sit for a few minutes to allow the protein to dissolve completely.

3. Further Dilution for Cell Culture

• Dilute the reconstituted stock in tissue culture medium or a buffered solution (e.g., PBS or serum-free medium) containing heat-inactivated fetal calf serum (FCS) or low endotoxin BSA (0.1–1%) to minimize protein loss and maintain stability.
• Typical working concentrations for cell culture assays range from 2.5–100 ng/mL, depending on the experimental design and cell type.

4. Storage

• Store reconstituted protein in aliquots at –20°C or –80°C to avoid repeated freeze-thaw cycles.
• If using a carrier protein, the protein is generally stable at 4°C for up to 1 week and at –20°C or –80°C for longer-term storage.

5. Handling Tips

• Use sterile, low-protein-binding tubes and pipette tips.
• Avoid vortexing or vigorous shaking to prevent protein denaturation.
• For cell culture, always filter-sterilize (0.22 µm) the final working solution if not already sterile.

Summary Table

Always refer to the specific product datasheet or Certificate of Analysis for exact instructions, as formulations may vary between suppliers.