Recombinant Rat Fractalkine (FKN)

Using Recombinant Rat Fractalkine (FKN, CX3CL1) in research applications is valuable for studying immune cell migration, inflammation, neurobiology, and metabolic regulation, as well as for modeling disease mechanisms and testing therapeutic interventions.

Key scientific reasons to use recombinant rat FKN include:

• Immune Cell Recruitment and Adhesion: FKN is a unique chemokine that exists in both membrane-bound and soluble forms. It mediates the adhesion and chemotaxis of T cells, monocytes, and NK cells to activated endothelium, making it essential for studying leukocyte trafficking and vascular inflammation.

• Inflammation and Disease Modeling: FKN plays a proinflammatory role in diseases such as rheumatoid arthritis by inducing synovial angiogenesis, activating monocytes and T cells, and stimulating matrix metalloproteinase production in synovial fibroblasts. It is also implicated in organ transplant rejection and fibrosis models, where the FKN–CX3CR1 axis regulates immune cell infiltration and tissue injury.

• Metabolic and Endocrine Research: FKN regulates insulin secretion and β-cell function. Chronic administration of FKN improves glucose tolerance, enhances insulin secretion, and reduces β-cell apoptosis in rodent models of obesity and diabetes, making it a tool for metabolic studies.

• Neurobiology: FKN is involved in neuron-glia communication and microglial regulation, relevant for neuroinflammation and neurodegenerative disease research. It is expressed on neurons and endothelial cells, and its receptor CX3CR1 is found on microglia and certain immune cells.

• Biomarker and Therapeutic Target Validation: FKN serves as a biomarker for disease prognosis (e.g., colorectal cancer) and as a target for therapeutic intervention in inflammatory and autoimmune diseases.

• In Vitro and In Vivo Functional Assays: Recombinant rat FKN is used to:

  • Chemoattract monocytes and lymphocytes in migration assays.
  • Stimulate or inhibit immune cell activation in co-culture or signaling studies.
  • Model receptor-ligand interactions with CX3CR1 in rodent systems, which is critical for translational research due to species-specific differences.

• High Purity and Defined Activity: Recombinant preparations offer high purity and consistent biological activity, essential for reproducible results in cell-based assays, ELISA standards, and mechanistic studies.

In summary, recombinant rat FKN is a versatile reagent for dissecting the roles of the FKN–CX3CR1 axis in immunity, inflammation, metabolism, and neurobiology, and for preclinical evaluation of potential therapeutic strategies targeting this pathway.

Yes, recombinant Rat Fractalkine (FKN, CX3CL1) can be used as a standard for quantification or calibration in ELISA assays, provided it is properly validated and matched to the assay system.

Supporting details:

• ELISA kits for rat Fractalkine routinely use recombinant protein as the standard to generate the calibration curve for quantification. The recombinant standard is serially diluted to create a standard curve, which is then used to determine the concentration of Fractalkine in unknown samples by comparison.
• Assay specificity and validation: Commercial ELISA kits are validated to recognize both natural and recombinant rat Fractalkine, ensuring that recombinant protein is suitable for use as a standard. The standard curve generated with recombinant protein is linear and accurate for quantification, as demonstrated in cytokine ELISA validation studies.
• Formulation considerations: Recombinant proteins may be supplied with or without carrier proteins (e.g., BSA). For ELISA standards, formulations with BSA are often recommended to improve stability and mimic sample matrix effects, but carrier-free protein may be preferred if BSA interferes with your assay.
• Sequence and domain matching: Ensure the recombinant protein used as a standard matches the relevant domain and sequence of the endogenous analyte detected by your assay antibodies. Most commercial recombinant rat Fractalkine proteins correspond to the soluble extracellular domain, which is typically the target of ELISA antibodies.

Best practices:

• Reconstitute and dilute the recombinant standard according to the ELISA kit or assay protocol, using the recommended buffer and diluent to ensure consistency and accuracy.
• Validate the standard curve in your assay system, especially if using a recombinant protein from a different source or with a different formulation than the kit standard.
• Store and handle the recombinant protein according to manufacturer recommendations to maintain activity and stability.

Summary: Recombinant Rat Fractalkine is widely accepted and validated as a standard for ELISA quantification, provided it matches the assay’s detection antibodies and is prepared according to protocol.

Recombinant Rat Fractalkine (FKN, CX3CL1) has been validated in published research for several key applications, primarily in neurobiology, immunology, and inflammation studies.

Validated Applications in Published Research:

• ELISA Standard and Recovery Control:
  Recombinant rat FKN is commonly used as a standard in ELISA assays to quantify endogenous fractalkine levels in biological samples. For example, it was used to assess recovery and quantification of soluble FKN in rat dorsal horn superfusates, demonstrating >80% recovery across a range of concentrations.

• Chemoattractant Assays:
  The biological activity of recombinant rat FKN has been validated by its ability to chemoattract human monocytes in vitro, typically at concentrations of 5–10 ng/mL, confirming its functional activity as a chemokine.

• Cell Culture and Functional Studies:
  Recombinant FKN is used in cell culture experiments to study its effects on immune cell migration, adhesion, and signaling, particularly in the context of neuron-glia interactions, microglial activation, and inflammatory responses.

• Neuroinflammation and Neuropathic Pain Models:
  In studies of neuropathic pain, recombinant rat FKN has been applied to dorsal horn preparations to investigate its release and role in microglial activation and pain signaling pathways.

• Rheumatic Disease and Arthritis Models:
  While most therapeutic studies focus on anti-FKN antibodies, recombinant FKN is used in preclinical models to elucidate its role in the pathogenesis of rheumatoid arthritis and related inflammatory conditions, particularly in the recruitment and activation of monocytes/macrophages.

Additional Context:

• ELISA and Immunoassays:
  Recombinant rat FKN is frequently used as a calibrator or positive control in ELISA kits designed to measure fractalkine in serum, plasma, or tissue culture supernatants.

• Mechanistic Studies:
  It is used to dissect the FKN–CX3CR1 signaling axis in various models, including studies of hippocampal neurogenesis, neuron-microglia communication, and inflammatory signaling in the central nervous system.

Summary Table: Applications of Recombinant Rat Fractalkine (FKN)

Note:
Most published research uses recombinant rat FKN for in vitro and ex vivo studies, including as a standard in immunoassays, a functional ligand in chemotaxis and signaling assays, and a tool to probe the FKN–CX3CR1 axis in disease models. It is not used therapeutically but rather as a research reagent to elucidate biological mechanisms.

To reconstitute and prepare Recombinant Rat Fractalkine (FKN/CX3CL1) protein for cell culture experiments, follow these best-practice steps:

1. Storage Before Use

• Store the lyophilized protein desiccated at -20°C or below until ready to use.

2. Preparation for Reconstitution

• Before opening, centrifuge the vial briefly to collect all powder at the bottom and minimize loss.

3. Reconstitution

• Buffer selection: Use sterile PBS (pH 7.4) or sterile distilled water for reconstitution, as recommended by most protocols.
• Concentration: Common reconstitution concentrations are 100 μg/mL in sterile PBS, or 0.1–1.0 mg/mL in sterile distilled water or PBS with 0.1% BSA (for stability).
• Procedure:
  • Add the appropriate volume of buffer directly to the vial to achieve the desired concentration.
  • Gently swirl or invert the vial to dissolve the protein. Avoid vigorous shaking or vortexing to prevent denaturation or foaming.
  • Allow the solution to sit at room temperature for 15–30 minutes to ensure complete dissolution.

4. Handling and Dilution

• For cell culture, further dilute the reconstituted protein in cell culture medium or buffer containing a carrier protein (e.g., 0.1% BSA, 10% FBS) to prevent adsorption to plastic and loss of activity, especially at low concentrations.
• If using in serum-free culture, avoid animal-derived carriers; instead, use a non-animal stabilizer like trehalose if needed.

5. Aliquoting and Storage

• Short-term storage: Store reconstituted protein at 2–8°C for up to 1 week.
• Long-term storage: Aliquot and freeze at -20°C to -80°C for several months. Avoid repeated freeze-thaw cycles to maintain activity.
• For long-term storage, adding carrier protein (e.g., 0.1% BSA) or glycerol (5–50%) can help stabilize the protein.

6. Usage in Experiments

• Thaw aliquots on ice and use immediately. Do not refreeze thawed aliquots.
• Typical working concentrations for chemotaxis or functional assays are in the 5–10 ng/mL range, but optimize based on your specific assay.

Summary Table: Key Steps

Critical Notes:

• Always consult the lot-specific Certificate of Analysis (CoA) for any manufacturer-specific instructions.
• Avoid repeated freeze-thaw cycles to preserve protein integrity.
• Use sterile technique throughout to prevent contamination.

These guidelines will ensure optimal activity and reproducibility of recombinant rat Fractalkine in cell culture experiments.