Recombinant Human CXCL16 (Extracellular Domain)

Recombinant Human CXCL16 (Extracellular Domain) is used in research applications to study its role as a chemokine involved in immune cell recruitment, extracellular matrix remodeling, and cell signaling, particularly in contexts such as cardiovascular disease, neurobiology, and inflammation.

CXCL16 exists as both a transmembrane protein and a soluble extracellular domain, with the latter functioning as a ligand for the CXCR6 receptor (also known as CD186, Bonzo, or STRL33). The recombinant extracellular domain allows for controlled in vitro and in vivo studies of CXCL16’s biological activities without interference from the transmembrane portion.

Key scientific applications include:

• Immune Cell Recruitment and Activation: CXCL16 is a potent recruiter of T cells and mononuclear cells, guiding their migration and accumulation in inflamed tissues such as rheumatoid arthritis synovium. It also promotes interactions between dendritic cells and CD8+ T cells, making it valuable for immunology studies.

• Extracellular Matrix Remodeling: CXCL16 induces matrix metalloproteinase (MMP) activity in cardiomyocytes and myocardial fibroblasts, attenuates collagen synthesis, and promotes fibroblast proliferation, implicating it in tissue remodeling and the pathogenesis of heart failure. Recombinant CXCL16 can be used to dissect these pathways in cell culture or animal models.

• Neurobiology: CXCL16 modulates neurotransmitter release at both inhibitory (GABAergic) and excitatory (glutamatergic) synapses, affecting synaptic transmission and plasticity in the hippocampus. The extracellular domain is essential for studying these effects in neuronal preparations.

• Cell Signaling Studies: The soluble extracellular domain of CXCL16 can activate signaling pathways (e.g., JNK, p38 MAPK, ERK) in target cells, enabling mechanistic studies of chemokine-receptor interactions and downstream effects.

• Apoptotic Cell Clearance: CXCL16 acts as a “find-me” signal for phagocytes by binding to phosphatidylserine on apoptotic bodies, facilitating their clearance.

• Therapeutic Target Validation: Recombinant CXCL16 is used to screen for inhibitors or antibodies that block its activity, which may have therapeutic potential in inflammatory diseases, cardiovascular disorders, and cancer.

Best practices for using recombinant CXCL16 (extracellular domain) include:

• Employing it in dose-response experiments to characterize cellular responses.
• Using it in chemotaxis assays to quantify immune cell migration.
• Applying it in signaling pathway analyses (e.g., Western blot, ELISA) to measure activation of downstream effectors.
• Incorporating it into co-culture systems to study cell-cell interactions mediated by CXCL16.

In summary, the recombinant extracellular domain of human CXCL16 is a versatile tool for dissecting its multifaceted roles in immunity, tissue remodeling, neurobiology, and cell signaling, providing precise control over experimental conditions and enabling mechanistic insights into CXCL16-mediated processes.

You can use recombinant human CXCL16 (extracellular domain) as a standard for quantification or calibration in your ELISA assays, provided that the recombinant protein is of high purity, its concentration is accurately determined, and it matches the form of CXCL16 recognized by your assay antibodies.

Key considerations and supporting details:

• Recombinant CXCL16 is commonly used as a standard in commercial ELISA kits. Many validated ELISA kits for human CXCL16 use recombinant CXCL16 (often the extracellular domain, expressed in E. coli or mammalian cells) as the calibration standard. These standards are used to generate the standard curve for quantification of CXCL16 in biological samples.

• The standard must match the assay’s antibody specificity. Ensure that the recombinant CXCL16 you use corresponds to the same region (extracellular domain) and isoform as the one recognized by the capture and detection antibodies in your ELISA. Most commercial kits are designed to detect both natural and recombinant forms of human CXCL16, provided the epitope is present.

• Concentration accuracy is critical. The recombinant protein must be accurately quantified (e.g., by absorbance at 280 nm, BCA assay, or amino acid analysis) to ensure reliable calibration. Impurities or inaccurate concentration determination will affect the accuracy of your standard curve.

• Matrix effects and recovery. When using recombinant CXCL16 as a standard, it is best to dilute it in a matrix similar to your samples (e.g., serum, plasma, or cell culture medium) to account for potential matrix effects. Many kits report good recovery and parallelism between recombinant and endogenous CXCL16 in various matrices.

• Documentation and validation. If you are developing your own ELISA or using a non-kit protocol, validate that your recombinant standard produces a standard curve with good linearity and that sample dilution curves are parallel to the standard curve, indicating comparable immunoreactivity.

Summary Table: Use of Recombinant CXCL16 as ELISA Standard

References to best practices:

• Commercial ELISA kits and development kits routinely use recombinant human CXCL16 as a standard and report parallelism with endogenous protein.
• Some manufacturers recommend using recombinant protein with carrier protein (e.g., BSA) for stability, especially for repeated freeze-thaw cycles.

Conclusion:
You can use recombinant human CXCL16 (extracellular domain) as a standard for ELISA quantification, provided it is well-characterized, matches the assay’s antibody specificity, and is validated for your specific assay conditions.

Recombinant Human CXCL16 (Extracellular Domain) has been validated for several applications in published research, primarily in functional cell-based assays, immunological studies, and biochemical analyses.

Key validated applications include:

• Functional Assays:
  Recombinant CXCL16 has been used to stimulate primary cells (e.g., myocardial fibroblasts, cardiomyocytes, breast cancer cells, neurons) to assess its effects on cell proliferation, migration, matrix metalloproteinase (MMP) activity, and signaling pathway activation (such as JNK, p38 MAPK, and ERK1/2). For example:

  • Induction of MMP activity and modulation of collagen synthesis in cardiac cells.
  • Modulation of cytoskeletal organization and cell motility in breast cancer models.
  • Regulation of neurotransmitter release in neuronal cultures.

• Immunological Studies:
  CXCL16 has been used to recruit and activate immune cells, particularly T cells, in both in vitro and in vivo models. For instance:

  • Recruitment of activated CD8⁺ T cells to tumors, enhancing antitumor immunity in mouse models.
  • Investigation of the CXCL16/CXCR6 axis in T cell infiltration and immune cell accumulation in tissues.

• ELISA (Enzyme-Linked Immunosorbent Assay):
  Recombinant CXCL16 is validated as a standard or control in ELISA for quantifying CXCL16 levels in biological samples.

• Biochemical and Signaling Studies:
  Used to probe downstream signaling events (e.g., phosphorylation of MAPK pathways) and to study receptor-ligand interactions, including the activation of CXCR6 and related pathways.

• Neuroscience Research:
  Application in studies of neurotransmitter release, where CXCL16 modulates synaptic activity in hippocampal neurons.

Summary Table of Validated Applications

Additional Notes:

• Most studies use the extracellular domain of CXCL16, as this is the biologically active, soluble form responsible for chemokine activity.
• Functional validation is typically performed using recombinant protein at nanomolar concentrations in cell culture or animal models.
• Some studies also use recombinant CXCL16 to explore its role in disease models, such as heart failure, cancer, and neuroinflammation.

If you require protocols or more specific details on assay conditions, please specify the intended application.

To reconstitute and prepare Recombinant Human CXCL16 (Extracellular Domain) protein for cell culture experiments, follow these best-practice steps based on current protocols and manufacturer recommendations:

1. Storage Before Reconstitution

• Store the lyophilized protein at –20 °C or lower, desiccated, until use.

2. Reconstitution

• Buffer: Use sterile phosphate-buffered saline (PBS). For increased stability and to prevent adsorption, add 0.1% human or bovine serum albumin (HSA/BSA) to the PBS.
• Concentration: Common reconstitution concentrations are 25–100 μg/mL. For example:
  • Reconstitute at 25 μg/mL in sterile PBS with 0.1% HSA/BSA.
  • Alternatively, reconstitute at 100 μg/mL in sterile PBS if higher stock concentration is desired.
• Procedure:
  • Briefly centrifuge the vial to collect all lyophilized material at the bottom.
  • Add the calculated volume of reconstitution buffer directly to the vial.
  • Gently swirl or invert (do not vortex) to dissolve. Allow to sit at room temperature for 10–30 minutes to ensure complete dissolution.

3. Aliquoting and Storage After Reconstitution

• Aliquot the reconstituted protein into small working volumes to avoid repeated freeze-thaw cycles.
• Store aliquots at –20 °C to –80 °C for long-term storage. For short-term use (up to 1 week), store at 2–8 °C.
• Avoid repeated freeze-thaw cycles, as this can denature the protein and reduce activity.

4. Preparation for Cell Culture

• Before adding to cell culture, dilute the stock solution to the desired working concentration using cell culture medium or PBS with 0.1% carrier protein (HSA/BSA) to minimize adsorption and maintain stability.
• Filter sterilize if necessary, especially if the buffer or handling conditions are not sterile.

5. Additional Notes

• Check the endotoxin level if using for sensitive cell types; most recombinant CXCL16 preparations are supplied with <1 EU/μg endotoxin.
• Always consult the specific product datasheet or certificate of analysis for any unique instructions related to your batch.

Summary Table: Key Steps for Recombinant Human CXCL16 (Extracellular Domain) Preparation

These steps will help ensure protein stability, activity, and reproducibility in your cell culture experiments.