Recombinant Human MIG (CXCL9)

Recombinant Human MIG (CXCL9) is widely used in research due to its critical roles in immune cell recruitment, modulation of immune responses, and potential as a biomarker and therapeutic target in cancer, inflammation, and immunotherapy studies.

Key reasons to use recombinant human CXCL9 in research applications include:

• Immune Cell Chemotaxis: CXCL9 is a potent chemoattractant for activated T lymphocytes (especially CD8+ and Th1 CD4+ T cells), NK cells, and other CXCR3-expressing cells, making it essential for studying immune cell migration and trafficking in vitro and in vivo.

• Tumor Immunology and Therapy: CXCL9 is highly relevant in cancer research. It can both promote antitumor immunity by recruiting effector T cells to the tumor microenvironment and, in some contexts, contribute to tumor progression by modulating the immune landscape. Its expression is associated with improved responses to immunotherapies, such as anti-PD-1/PD-L1 treatments, and can serve as a prognostic or predictive biomarker in several cancers.

• Angiostasis: CXCL9, along with related chemokines, exhibits angiostatic properties, inhibiting the formation of new blood vessels in tumors and inflamed tissues, which is valuable for studying tumor angiogenesis and anti-angiogenic therapies.

• Inflammation and Autoimmunity: CXCL9 is upregulated in response to interferon-gamma and is involved in the pathogenesis of various inflammatory and autoimmune diseases, making it a useful tool for modeling and dissecting these conditions.

• Bioassays and Functional Studies: Recombinant CXCL9 is used in chemotaxis assays, cytokine production studies, and functional immune assays to elucidate mechanisms of immune cell activation, migration, and effector function.

• Vaccine and Immunotherapy Research: By enhancing immune cell recruitment, CXCL9 can be leveraged to improve vaccine efficacy and the effectiveness of adoptive cell therapies, such as CAR T cells, by promoting immune infiltration into target tissues or tumors.

• Standardization and Controls: Recombinant CXCL9 serves as a standard in ELISA, Western blot, and other immunoassays, ensuring reproducibility and quantification in experimental protocols.

In summary, recombinant human CXCL9 is a versatile reagent for dissecting immune mechanisms, modeling disease, developing diagnostics, and testing therapeutic strategies in immunology, oncology, and inflammation research.

Yes, recombinant human MIG (CXCL9) can be used as a standard for quantification or calibration in ELISA assays, provided it is of high purity and properly validated for this application.

Supporting details and best practices:

• Recombinant human MIG (CXCL9) is commonly used as the standard in commercial ELISA kits. These kits typically use E. coli-expressed recombinant human MIG as the calibrator, and validation data show that the standard curves generated with recombinant MIG are parallel to those generated with natural human MIG, indicating equivalence for quantification purposes.
• The standard should be reconstituted and diluted according to the ELISA kit protocol to generate a standard curve covering the assay’s dynamic range. For example, a typical protocol involves reconstituting the lyophilized recombinant MIG to a known concentration (e.g., 20,000 pg/mL), then preparing serial dilutions to create the standard curve.
• Recovery and linearity studies in validated kits demonstrate that recombinant MIG is accurately quantified in various matrices (serum, plasma, cell culture supernatant), with recoveries typically between 92–109% and linearity across the assay range.
• Specificity data confirm that the antibodies used in these ELISAs recognize both recombinant and natural human MIG, and there is no significant cross-reactivity with related chemokines.
• For best results, ensure your recombinant MIG is:
  • Highly purified (typically >98% by SDS-PAGE).
  • Free of significant endotoxin contamination if using in cell-based assays.
  • Matched in sequence and post-translational modifications (if relevant) to the native protein detected by your assay antibodies.

Caveats:

• Use a new standard preparation for each assay to ensure accuracy and reproducibility.
• If using a recombinant MIG standard from a source different from your ELISA kit, confirm that it is compatible with your assay’s antibodies and detection system. Some vendors note that their recombinant proteins are not validated for use as ELISA standards unless specifically stated.
• Always follow the specific instructions provided in your ELISA kit manual for standard preparation and calibration.

Summary:
Recombinant human MIG (CXCL9) is widely accepted and validated as a standard for ELISA quantification, provided it is of high quality and used according to established protocols.

Recombinant Human MIG (CXCL9) has been validated for several key applications in published research, primarily in studies of immune cell chemotaxis, tumor immunology, and as a biomarker for immune activation.

Validated Applications:

• Cell Migration/Chemotaxis Assays: CXCL9 is widely used to study the chemotactic recruitment of activated T cells, tumor-infiltrating lymphocytes (TILs), and NK cells via CXCR3 signaling. These assays typically involve transwell migration or Boyden chamber setups to quantify cell movement in response to recombinant CXCL9.

• Tumor Immunology and Immunotherapy Models: Recombinant CXCL9 has been used to manipulate the tumor microenvironment, increasing infiltration of cytotoxic lymphocytes and sensitizing tumors (e.g., glioblastoma, ovarian cancer) to immune checkpoint blockade therapies such as anti-PD-1 or anti-PD-L1. These studies often involve in vivo administration or gene therapy approaches to deliver CXCL9 and assess therapeutic outcomes.

• Functional Biomarker Assays: CXCL9 secretion is a sensitive measure of bioactive IFN-γ signaling and Th1-type immune responses. It has been used in flow cytometry, ELISA, and RT-PCR assays to monitor immune activation post-vaccination or infection, often providing greater sensitivity than IFN-γ detection alone.

• ELISA Standard and Western Blot Control: Recombinant CXCL9 serves as a standard for quantification in ELISA assays and as a positive control in Western blotting, enabling accurate measurement of endogenous CXCL9 levels in biological samples.

• Cell Culture Supplementation: It is used to stimulate or modulate immune cell cultures, particularly to induce chemotactic responses or to study downstream signaling pathways activated by CXCR3 engagement.

Additional Context:

• CXCL9 is not typically a chemoattractant for neutrophils or monocytes, but specifically targets activated T cells and TILs.
• Its role as a biomarker extends to predicting immunotherapy efficacy and monitoring immune responses in cancer and infectious disease models.
• Recombinant CXCL9 is for research use only and not intended for diagnostic or therapeutic applications in humans or animals.

Summary Table:

These applications are supported by peer-reviewed studies and product documentation, reflecting the broad utility of recombinant human CXCL9 in immunological research.

To reconstitute and prepare Recombinant Human MIG (CXCL9) protein for cell culture experiments, dissolve the lyophilized protein in sterile PBS or sterile water to a concentration of 100 μg/mL (or as recommended by the supplier), using gentle pipetting and avoiding vortexing. For optimal stability and activity, include a carrier protein such as 0.1–1% BSA if the protocol or application requires it.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to collect the powder at the bottom.
• Add sterile PBS or sterile water to achieve the desired concentration (commonly 100 μg/mL for stock solutions).
• Gently pipette the solution down the sides of the vial to dissolve the protein. Do not vortex, as this may denature the protein.
• Allow several minutes for complete reconstitution at room temperature.
• If required for stability or to prevent adsorption, add 0.1–1% BSA or HSA to the buffer.
• Aliquot the stock solution to avoid repeated freeze-thaw cycles.
• Store aliquots at −20°C or −80°C for long-term storage; short-term storage (up to one week) can be at 2–8°C.
• For cell culture use, dilute the stock solution into your culture medium to the desired working concentration immediately before use.

Additional notes:

• Avoid repeated freeze-thaw cycles to preserve protein integrity.
• If the protein is formulated with additives (e.g., trehalose, TFA), ensure compatibility with your cell culture system.
• For sensitive applications, confirm endotoxin levels are suitable for cell culture (<1 EU/μg is typical).
• If using for ELISA or as a standard, follow the specific diluent recommendations (e.g., Sample Diluent NS or 0.1% BSA in PBS).

Summary Table:

This protocol ensures the recombinant CXCL9/MIG protein is properly reconstituted and prepared for cell culture experiments, maintaining its biological activity and stability.