Recombinant Mouse KC/CXCL1

Recombinant Mouse KC/CXCL1 is widely used in research because it is a key chemokine involved in neutrophil recruitment, inflammation, angiogenesis, and immune cell modulation in mouse models. Its recombinant form allows for precise, reproducible studies of its biological functions and signaling pathways.

Key applications and scientific rationale include:

• Neutrophil Chemotaxis and Inflammation: CXCL1/KC is a potent chemoattractant for neutrophils, mediating their migration, extravasation, and activation during acute and chronic inflammatory responses. This makes it essential for studying innate immune mechanisms, infection models, and inflammatory diseases.

• Angiogenesis and Tissue Remodeling: CXCL1 promotes endothelial cell migration and tube formation, contributing to angiogenesis and tissue repair. It also influences chondrocyte differentiation and cartilage matrix remodeling, relevant for studies in tissue engineering and regenerative medicine.

• Tumor Biology: CXCL1 is implicated in tumor progression, angiogenesis, and metastasis by modulating the tumor microenvironment and recruiting immune and stromal cells. Its use is critical in cancer research to dissect chemokine-driven mechanisms of tumor growth and immune evasion.

• Neuroinflammation and CNS Disease: In models of neuroinflammatory diseases, such as demyelinating disorders, CXCL1 regulates leukocyte infiltration and T cell responses in the central nervous system. Recombinant CXCL1 enables controlled studies of neuroimmune interactions.

• Immune Cell Modulation: CXCL1 not only attracts neutrophils but also induces T cells to produce proinflammatory cytokines like IL-17, influencing both innate and adaptive immunity.

• In Vivo and In Vitro Assays: Recombinant CXCL1 is used in bioassays, chemotaxis assays, in vivo inflammation models, and binding studies to elucidate receptor interactions (primarily CXCR2, with lower affinity for CXCR1).

• Disease Modeling: It is used to model and manipulate inflammatory, autoimmune, cardiovascular, and neurodegenerative diseases in mice, providing insights into pathophysiology and therapeutic targeting.

Using recombinant protein ensures batch-to-batch consistency, defined activity, and species specificity, which are critical for reproducible and interpretable experimental results. This is especially important when dissecting chemokine signaling pathways or evaluating therapeutic interventions targeting the CXCL1/CXCR2 axis.

Yes, recombinant Mouse KC/CXCL1 can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and properly calibrated. Recombinant mouse KC/CXCL1 is commonly used as a standard in commercial ELISA kits, and its dose-response curves are parallel to those obtained with natural mouse KC, allowing for accurate quantification.

Supporting details:

• Parallelism and Calibration: ELISA kits designed for mouse KC/CXCL1 typically use E. coli-expressed recombinant mouse KC as the standard. Studies and kit documentation confirm that the standard curves generated with recombinant KC are parallel to those obtained with natural KC, indicating that recombinant KC is suitable for determining relative mass values of natural KC in samples.

• Assay Principle: The quantification is based on a sandwich ELISA format, where the intensity of the colorimetric signal is directly proportional to the concentration of KC/CXCL1 in the sample. The recombinant protein standard is serially diluted to generate a standard curve, which is then used to interpolate the concentration of KC/CXCL1 in unknown samples.

• Purity and Suitability: Recombinant KC/CXCL1 used as a standard should be highly purified (typically >97–98% by SDS-PAGE) and free of significant endotoxin contamination to ensure accuracy and reproducibility. It is important to use recombinant protein preparations that are specifically validated for ELISA standard use, as not all recombinant proteins are tested for this application.

• Limitations: The recombinant standard should be matched to the ELISA kit’s specifications, and care should be taken not to mix reagents from different lots or manufacturers, as this can affect assay performance. The use of recombinant KC/CXCL1 as a standard is for research purposes only and not for diagnostic procedures.

Best practices:

• Prepare a dilution series of the recombinant KC/CXCL1 according to the ELISA kit protocol to generate a standard curve.
• Confirm that the recombinant protein is validated for use as an ELISA standard and matches the kit’s requirements for purity and formulation.
• Ensure all reagents and standards are handled according to the manufacturer’s instructions to maintain assay accuracy.

Summary Table: Recombinant Mouse KC/CXCL1 as ELISA Standard

In conclusion, recombinant Mouse KC/CXCL1 is widely accepted and validated as a standard for ELISA quantification and calibration, provided it meets purity and validation criteria and is used according to established protocols.

Recombinant Mouse KC/CXCL1 has been validated for a wide range of applications in published research, primarily focusing on its roles in inflammation, immune cell recruitment, and disease models. The main applications include:

• In Vivo Studies:

  • Neutrophil migration and recruitment to inflammatory sites
  • Murine sickle cell vaso-occlusion during hemolytic transfusion reactions
  • Promotion of lymphatic tube formation and angiogenesis
  • Cartilage matrix deposition, calcification, and remodeling
  • Recruitment of adipose stromal cells to tumors in obese models
  • Ovarian cancer cell proliferation via EGF receptor transactivation

• Bioassays:

  • Chemotaxis assays using neutrophils or Baf3-mCXCR2 transfectants (dose-dependent chemoattraction, with ED50 values reported)
  • Measurement of neutrophil mobilization from bone marrow in acute peritonitis models
  • Assessment of cell proliferation, tumor angiogenesis, invasion, and metastasis

• In Vitro Cell Culture:

  • Induction of neutrophil migration and activation
  • Endothelial cell migration and tube formation assays
  • Stimulation of osteoclast precursor recruitment in osteoblast cultures
  • Evaluation of cytokine-induced expression (e.g., by IL-1, TNF, thrombin, PGE2)

• Disease Biomarker Discovery:

  • Identification as a diagnostic biomarker for active tuberculosis in mouse and human studies
  • Use in panels for discriminating active TB from latent TB and non-TB lung disease

• Functional Studies:

  • Investigation of CXCR2 signaling and receptor-ligand interactions
  • Assessment of chemokine-induced respiratory burst and degranulation in neutrophils
  • Analysis of T cell IL-17 production in response to CXCL1

These applications highlight the utility of Recombinant Mouse KC/CXCL1 in both basic research and translational studies involving inflammation, immunity, cancer, and infectious disease.

To reconstitute and prepare Recombinant Mouse KC/CXCL1 protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer to a concentration of 0.1–1.0 mg/mL, avoiding vigorous mixing such as vortexing. For most cell culture applications, reconstitution in sterile PBS (phosphate-buffered saline) at 50–100 μg/mL is common, and adding a carrier protein such as 0.1–1% BSA (bovine serum albumin) or HSA (human serum albumin) is recommended to stabilize the protein and minimize adsorption to plastic surfaces.

Step-by-step protocol:

1. Briefly centrifuge the vial to collect all lyophilized material at the bottom before opening.
2. Add sterile water or PBS:
   • For general use, add sterile water or PBS to achieve a final concentration of 0.1–1.0 mg/mL.
   • For cell culture, 50–100 μg/mL in sterile PBS is typical.
3. Add carrier protein (optional but recommended for stability):
   • Add 0.1–1% BSA or HSA to the buffer if the protein will be stored or diluted further.
4. Gently mix by pipetting up and down; do not vortex.
5. Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.
6. Storage:
   • Short-term (up to 1 week): 2–8°C.
   • Long-term: -20°C or -80°C in working aliquots, especially if diluted in buffer with carrier protein.
   • Avoid repeated freeze/thaw cycles to preserve activity.

Additional notes:

• For bioassays, working concentrations are typically in the range of 0.1–10 ng/mL, but optimal dosing should be determined empirically for your specific cell type and assay.
• After reconstitution, confirm protein integrity by SDS-PAGE if needed.
• If using a carrier-free formulation, extra care should be taken to prevent protein loss due to adsorption; always use low-protein-binding tubes and add carrier protein if possible.

Summary Table:

This protocol ensures maximal stability and biological activity of recombinant mouse KC/CXCL1 for cell culture experiments.