Recombinant Human CXCL14

Using Recombinant Human CXCL14 in research applications is valuable for studying its roles in immune regulation, tumor suppression, and tissue repair, as well as for developing novel immunotherapeutic strategies.

Key scientific reasons to use recombinant human CXCL14 include:

• Antitumor Immunity: CXCL14 has been shown to promote antitumor immune responses by recruiting and activating CD8⁺ T cells and natural killer (NK) cells within the tumor microenvironment (TME). It also restores or upregulates MHC-I expression on tumor cells, enhancing antigen presentation and facilitating immune-mediated tumor cell killing.

• Immunotherapy Research: High CXCL14 expression correlates with improved patient survival in several cancers, including HPV-positive head and neck squamous cell carcinoma (HNSCC) and cervical cancer. Recombinant CXCL14 is being explored as an immunotherapeutic agent, particularly in combination with immune checkpoint inhibitors (ICIs), to boost responses in patients who do not respond to standard immunotherapies.

• Tissue Repair and Regeneration: Recombinant CXCL14 accelerates corneal wound healing by promoting the proliferation, migration, and stemness of corneal epithelial cells, highlighting its therapeutic potential in tissue repair models.

• Immune Cell Recruitment: CXCL14 acts as a chemoattractant for dendritic cells, monocytes, and macrophage precursors, making it useful for studying immune cell trafficking and the regulation of local immune responses in various tissues.

• Functional Assays and Mechanistic Studies: Recombinant CXCL14 is suitable for use in functional assays (e.g., chemotaxis, cell migration, immune activation), ELISA, and mechanistic studies to dissect its signaling pathways and biological effects.

• Modeling Disease Mechanisms: Loss or downregulation of CXCL14 is implicated in cancer progression and immune evasion, so recombinant protein can be used to model disease mechanisms and test therapeutic hypotheses in vitro and in vivo.

In summary, recombinant human CXCL14 is a versatile tool for investigating immune modulation, cancer biology, tissue regeneration, and for preclinical evaluation of new therapeutic strategies targeting the CXCL14 axis.

You can use recombinant human CXCL14 as a standard for quantification or calibration in your ELISA assays, provided it is of high purity, accurately quantified, and compatible with your assay system.

Key considerations:

• Source and Purity: The recombinant CXCL14 should be well-characterized, with known purity and concentration. Carrier-free forms are preferred for ELISA calibration to avoid interference from stabilizers like BSA unless your assay specifically requires a carrier protein.
• Compatibility: Ensure the recombinant CXCL14 matches the sequence and post-translational modifications (if relevant) of the endogenous protein detected by your ELISA antibodies. Most commercial ELISA kits use recombinant CXCL14 as their standard, typically produced in E. coli or mammalian systems.
• Standard Curve Preparation: Reconstitute the recombinant protein according to the manufacturer’s instructions, dilute in the same buffer as your samples, and prepare a standard curve covering the assay’s dynamic range. The standard curve should be freshly prepared for each assay run to ensure accuracy.
• Validation: Confirm that the recombinant standard yields a linear, reproducible standard curve and that its detection is equivalent to native CXCL14 in your sample matrix. This is standard practice in ELISA development and is supported by protocols from major kit manufacturers.

Best practices:

• Use the same diluent for standards and samples to minimize matrix effects.
• Validate recovery and parallelism by spiking recombinant CXCL14 into your sample matrix.
• Store aliquots of the recombinant standard at −20°C or as recommended to maintain stability.

Summary:
Recombinant human CXCL14 is widely used as a standard in quantitative ELISA assays, provided it is properly validated for your specific assay conditions. Always follow best practices for standard preparation and assay validation to ensure accurate quantification.

Recombinant Human CXCL14 has been validated for several applications in published research, including functional assays, ELISA, Western blot, and various in vitro and in vivo bioassays.

Key validated applications and research uses include:

• Functional Assays: Recombinant CXCL14 is widely used in cell-based functional assays to study its effects on cell migration, proliferation, and signaling pathways. For example, it has been used to assess chemotactic activity for immune cells such as monocytes and dendritic cells.

• ELISA (Enzyme-Linked Immunosorbent Assay): Used as a standard or control protein to quantify CXCL14 levels in biological samples.

• Western Blot: Applied as a positive control or for detection of CXCL14 in protein extracts, including studies of signaling pathway activation (e.g., NF-κB, AKT-mTOR).

• Bioassays and Mechanistic Studies:

  • Tumor Immunology: Recombinant CXCL14 has been used to demonstrate its role in recruiting and activating CD8+ T cells, restoring MHC-I expression, and suppressing tumor growth in HPV-positive head and neck cancer models.
  • Tissue Repair: It has been validated in corneal wound healing models, where it promotes proliferation, migration, and stemness of corneal epithelial cells via NF-κB signaling.
  • Muscle Biology: Used in studies showing that CXCL14 reverses muscle atrophy and regulates protein synthesis/degradation pathways in myotubes and muscle tissue, acting through the AKT-S6K and FOXO pathways.
  • Trophoblast Invasion: Applied in assays demonstrating its ability to restrict human trophoblast cell invasion by suppressing gelatinase activity.

• In Vivo Models: Recombinant CXCL14 has been administered in animal models (e.g., rat corneal injury, mouse muscle atrophy) to validate its therapeutic and biological effects.

Summary Table of Validated Applications

These applications are supported by both vendor validation and peer-reviewed studies, demonstrating the protein’s utility in immunology, oncology, regenerative medicine, and cell signaling research.

To reconstitute and prepare Recombinant Human CXCL14 protein for cell culture experiments, dissolve the lyophilized protein in sterile, high-purity water (18 MΩ·cm) at a concentration of not less than 100 μg/mL. After reconstitution, further dilute the stock solution to your desired working concentration using sterile buffer or cell culture medium.

Step-by-step protocol:

1. Centrifuge the vial briefly to ensure all lyophilized protein is at the bottom before opening.
2. Add sterile water: Use sterile, endotoxin-free water (18 MΩ·cm) to reconstitute the protein. For most protocols, a concentration of ≥100 μg/mL is recommended for initial stock preparation.
3. Gently mix: Do not vortex or pipette vigorously. Allow the protein to dissolve by gentle inversion or by letting it sit at room temperature for several minutes.
4. Aliquot: Divide the reconstituted stock into small aliquots to avoid repeated freeze-thaw cycles.
5. Storage:
   • Store aliquots at -20°C to -80°C for long-term use.
   • For short-term use (up to 1 month), aliquots can be kept at 2–8°C.
   • Avoid repeated freeze-thaw cycles, as this can reduce protein activity.
6. Dilution for cell culture:
   • Dilute the stock solution to the desired working concentration using sterile cell culture medium or buffer.
   • If stability is a concern, add a carrier protein such as 0.1–1% BSA or HSA to the working solution.
   • Typical working concentrations for bioassays range from 1.5–7.5 μg/mL, but optimize based on your experimental needs.

Additional notes:

• Always consult the specific product datasheet for any unique instructions regarding buffer composition, pH, or additives (e.g., trehalose, mannitol, Tween 80).
• If the protein is supplied in solution, it may already be in PBS or another buffer and can be used directly after dilution.
• For cell culture, ensure all solutions are sterile and endotoxin-free to prevent confounding effects.

Summary Table:

This protocol ensures optimal solubility, stability, and biological activity of recombinant CXCL14 for cell culture experiments.