Recombinant Mouse C-X-C motif chemokine 10 protein (Cxcl10)

What is the basic structure of recombinant mouse CXCL10 protein?

Recombinant Mouse CXCL10 (IP-10) is a single, non-glycosylated polypeptide chain containing 77 amino acids with a molecular mass of approximately 8701 Daltons. The protein's N-terminal sequence begins with Ile-Pro-Leu-Ala-Arg. The three-dimensional crystal structure of this chemokine has been determined under three different conditions to a resolution of up to 1.92Å. When expressed in E. coli systems, the protein maintains its monomeric structure and is typically purified using proprietary chromatographic techniques to achieve greater than 97% purity as determined by RP-HPLC and SDS-PAGE analysis .

How does mouse CXCL10 compare to human CXCL10 in terms of structure and function?

While the search results don't provide a direct comparison, both mouse and human CXCL10 belong to the CXC chemokine family and function as pro-inflammatory cytokines that bind to the CXCR3 receptor. Both are involved in chemotaxis, cellular differentiation, and activation of immune cells. The mouse CXCL10 protein sequence (I P L A R T V R C N C I H I D D G P V R M R A I G K L E I I P A S L S C P R V E I I A T M K K N D E Q R C L N P E S K T I K N L M K A F S Q K R S K R A P) shares significant homology with human CXCL10, though species-specific differences in potency and receptor binding affinity exist . Researchers should be aware of these differences when designing cross-species studies or translating findings from mouse models to human applications.

What are the optimal storage and reconstitution conditions for recombinant mouse CXCL10?

Lyophilized mouse CXCL10 should be stored desiccated below -18°C, though it remains stable at room temperature for approximately three weeks. For reconstitution, it is recommended to use sterile 18MΩ-cm H₂O at a concentration not less than 100μg/ml, which can then be further diluted to other aqueous solutions. After reconstitution, CXCL10 should be stored at 4°C if used within 2-7 days. For longer-term storage (beyond 7 days), the reconstituted protein should be stored below -18°C with the addition of a carrier protein (0.1% HSA or BSA) to enhance stability. Multiple freeze-thaw cycles should be strictly avoided as they can significantly compromise protein integrity and activity .

How is the biological activity of recombinant mouse CXCL10 measured in research settings?

The biological activity of recombinant mouse CXCL10 is primarily quantified by its ability to chemoattract IL-2 activated T cells, with effective concentrations typically ranging from 0.1-10 ng/ml. This chemotactic assay serves as the gold standard for determining CXCL10 functionality. Additionally, researchers can measure CXCL10 activity through its ability to activate signaling pathways upon binding to the CXCR3 receptor, particularly through G protein-mediated signaling resulting in phospholipase C activation, increased intracellular calcium production, and actin reorganization. More specialized assays may include measuring its ability to activate Erk and p38-MAPK signaling pathways in target cells such as macrophages, which can be detected through phosphorylation-specific antibodies in Western blot analyses .

What are effective approaches for studying CXCL10-mediated signaling pathways in vitro?

To study CXCL10-mediated signaling pathways in vitro, researchers can employ several methodological approaches:

• Receptor inhibition studies: Pretreating cells with CXCR3 antagonists like AMG487 (1 μM) for 1 hour before CXCL10 stimulation (typically 100 ng/ml) allows researchers to confirm pathway specificity.

• Phosphorylation analysis: Western blotting to detect phosphorylated forms of Erk and p38-MAPK at various time points (5-60 minutes) after CXCL10 stimulation provides insight into activation kinetics.

• Gene expression profiling: RT-qPCR analysis of cells following CXCL10 stimulation (100 ng/ml for 12 hours) helps identify downstream transcriptional changes.

• Combined stimulation models: Pretreating bone marrow-derived macrophages (BMDMs) with CXCL10 (100 ng/ml) for 12 hours followed by LPS stimulation (50 ng/ml for 3 hours) enables the study of CXCL10's modulatory effects on other inflammatory pathways .

These approaches allow for comprehensive characterization of the signaling cascades activated by CXCL10 binding to CXCR3 receptors in various cell types.

How can CXCL10 be effectively silenced in mouse models for functional studies?

For in vivo CXCL10 silencing, short hairpin RNA (shRNA) delivered via adeno-associated virus (AAV) vectors has proven effective. The target sequence 5′-TTGATGGTCTTAGATTCCGGA-3′ has been validated in previous research for specific CXCL10 knockdown. When packaging this shRNA into AAV vectors, titers of approximately 1.6 × 10^10 viral particles/ml are typically used to ensure sufficient transduction efficiency. Control experiments should include the same vector containing an empty vector backbone (AAV-control) administered at equivalent titers. This approach allows for tissue-specific CXCL10 silencing without affecting other chemokine pathways, enabling researchers to investigate the specific contributions of CXCL10 to inflammation, immune cell recruitment, and disease pathogenesis in various experimental models .

How does CXCL10 contribute to neuroinflammatory processes and what methods are optimal for studying these mechanisms?

CXCL10 plays a crucial role in neuroinflammatory processes, particularly in response to brain injury. The CXCL10/CXCR3 axis activates microglia, the resident macrophage population of the central nervous system, and directs them to lesion sites. This recruitment process is essential for neuronal reorganization following injury. To study these mechanisms, researchers can employ several specialized approaches:

• Ex vivo slice cultures: Brain slice cultures treated with recombinant CXCL10 can reveal microglial activation and migration patterns using time-lapse microscopy.

• In vivo models: Stereotactic injection of CXCL10 into specific brain regions followed by immunohistochemical analysis of microglial markers (Iba1, CD68) can demonstrate the protein's chemotactic effects.

• CXCR3 conditional knockout models: Cell-specific CXCR3 deletion in microglia versus neurons helps distinguish direct versus indirect effects of CXCL10 signaling in neuroinflammation.

• Cytokine profiling: Multiplex cytokine assays of brain tissue or cerebrospinal fluid following CXCL10 administration can reveal the broader inflammatory consequences of CXCL10-mediated microglial activation .

Understanding these mechanisms has significant implications for neurological disorders characterized by inflammation, including traumatic brain injury, stroke, and neurodegenerative diseases.

What is the role of CXCL10 in autoimmune disease models and how can it be therapeutically targeted?

CXCL10 plays significant pathogenic roles in autoimmune disease models, particularly in experimental autoimmune prostatitis. High CXCL10 expression correlates with disease severity and pain symptoms. Mechanistically, CXCL10 induces proinflammatory factor secretion and macrophage chemotaxis through activation of Erk and p38-MAPK signaling pathways via CXCR3 binding. This leads to enhanced inflammatory cell accumulation and cytokine production.

For therapeutic targeting, several approaches have shown promise:

• Direct CXCL10 neutralization: Using anti-CXCL10 antibodies to neutralize the protein in vivo.

• CXCR3 antagonism: Small molecule inhibitors of CXCR3 (such as AMG487) can block CXCL10 signaling at the receptor level.

• Gene silencing: AAV-delivered shRNA targeting CXCL10 has demonstrated effectiveness in reducing disease severity and inflammatory infiltration in mouse models.

• Upstream regulation: Targeting the pathways that induce CXCL10 expression, particularly interferon-gamma signaling.

Each approach offers distinct advantages and limitations, with CXCR3 antagonism currently showing the most translational potential due to the availability of small molecule inhibitors with favorable pharmacokinetic profiles .

How can CXCL10 be used as a biomarker in infectious disease research?

CXCL10 shows promise as a biomarker in infectious disease research, particularly in tuberculosis (TB) treatment monitoring. While the search results provide limited details on this application, evidence suggests that CXCL10, along with CXCL9, may serve as indicators of treatment efficacy in TB patients. The advantages of using CXCL10 as a biomarker include:

• Non-invasive assessment: CXCL10 can be measured in peripheral blood, making it more accessible than traditional methods requiring sputum samples.

• Early response indicator: Changes in CXCL10 levels may precede clinical improvement or bacterial clearance, allowing for earlier assessment of treatment efficacy.

• Discrimination potential: CXCL10 levels might help distinguish between active disease, latent infection, and successful treatment.

• Correlation with inflammatory load: As a pro-inflammatory chemokine, CXCL10 levels correlate with the degree of inflammation, potentially reflecting disease activity.

Researchers investigating CXCL10 as a biomarker should consider longitudinal sampling, correlation with other established biomarkers, and integration with clinical parameters to maximize its utility in monitoring treatment responses .

What are common pitfalls in working with recombinant CXCL10 and how can they be addressed?

When working with recombinant mouse CXCL10, researchers commonly encounter several technical challenges:

• Protein aggregation: CXCL10 can form aggregates after reconstitution, particularly at high concentrations. This can be minimized by reconstituting the lyophilized protein slowly at room temperature with gentle agitation rather than vortexing, and by adding a carrier protein (0.1% HSA or BSA) for stabilization.

• Activity loss from freeze-thaw cycles: Multiple freeze-thaw cycles significantly reduce biological activity. Researchers should aliquot the reconstituted protein for single use and store at -80°C.

• Adsorption to plastics: CXCL10 may adsorb to plastic surfaces during storage or experimental procedures. Using low-binding microcentrifuge tubes and pipette tips, and including 0.1% BSA in buffers can reduce protein loss.

• Endotoxin contamination: When using CXCL10 in cell-based assays, endotoxin contamination can confound results. Source CXCL10 preparations with certified low endotoxin levels (≤0.005 EU/μg) and consider including polymyxin B controls in sensitive assays to rule out endotoxin effects .

• Receptor desensitization: Prolonged exposure to CXCL10 can cause CXCR3 receptor desensitization. Design time-course experiments carefully and consider including receptor recycling time in protocols.

How should researchers validate the activity and specificity of recombinant mouse CXCL10 in their experimental systems?

Thorough validation of recombinant mouse CXCL10 activity and specificity is crucial for reliable experimental outcomes. A comprehensive validation approach should include:

• Functional chemotaxis assay: Verify chemotactic activity using IL-2 activated T cells at concentrations ranging from 0.1-10 ng/ml. A proper dose-response curve should be established to determine the optimal working concentration for each experimental system.

• Receptor binding specificity: Confirm CXCR3-specific effects by comparing responses in CXCR3-positive versus CXCR3-negative cell lines, or by using CXCR3 antagonists like AMG487 as controls.

• Signaling pathway activation: Verify activation of canonical signaling pathways using phospho-specific antibodies against Erk and p38-MAPK in Western blots. Activation should be dose-dependent and follow expected kinetics.

• Biological response verification: Confirm that treatment induces expected biological responses (e.g., cytokine production, gene expression changes) in relevant cell types such as macrophages and T cells.

• Cross-reactivity testing: If working with mixed species systems, verify the species-specificity of your recombinant mouse CXCL10 to avoid misinterpretation of cross-species effects .

This systematic validation approach helps ensure that observed effects are specifically attributable to CXCL10 activity rather than contaminants or non-specific interactions.

What quantification methods are most appropriate for measuring CXCL10 in different experimental contexts?

The appropriate method for CXCL10 quantification depends on the experimental context and sample type. Based on the search results and scientific practice, recommended approaches include:

• Protein content in recombinant preparations:

  • UV spectroscopy at 280 nm using an extinction coefficient of 0.02 for a 0.1% (1mg/ml) solution

  • RP-HPLC analysis against a reference standard of known concentration

  • These methods are particularly suitable for quality control of recombinant preparations

• CXCL10 in biological samples:

  • Enzyme-linked immunosorbent assay (ELISA) for precise quantification in serum, plasma, or tissue culture supernatants

  • Multiplex bead-based assays for simultaneous measurement of CXCL10 alongside other cytokines/chemokines

  • Western blotting for semi-quantitative assessment in tissue or cell lysates

• Gene expression analysis:

  • RT-qPCR for measuring CXCL10 mRNA expression in cells or tissues

  • RNAscope in situ hybridization for localization and quantification in tissue sections

• Single-cell analysis:

  • Flow cytometry with intracellular cytokine staining for identifying CXCL10-producing cells

  • Single-cell RNA sequencing for comprehensive profiling of CXCL10 expression at the single-cell level

The choice between these methods should be guided by the specific research question, required sensitivity, sample volume constraints, and the need for absolute versus relative quantification.

How is CXCL10 being investigated in neurological disease models beyond its basic chemotactic functions?

Beyond its established role in chemotaxis, CXCL10 is being investigated for its broader impacts in neurological disease models. Recent research indicates that CXCL10/CXCR3 signaling plays critical roles in neuronal reorganization following brain injury. This process involves not just the recruitment of microglia to lesion sites but also direct effects on neural cells. The neuronal response to CXCL10 appears to be essential for effective repair and adaptation following injury.

Emerging research directions include:

• Neuronal-glial interactions: Investigating how CXCL10-mediated signaling facilitates communication between neurons and glia during injury response.

• Synaptic plasticity: Examining CXCL10's potential roles in modulating synaptic strength and neuronal connectivity through CXCR3 signaling.

• Neurogenesis and neural stem cell function: Exploring how CXCL10 influences neural stem cell proliferation, differentiation, and integration in injury contexts.

• Neuroprotective versus neurotoxic effects: Determining the concentration-dependent and context-specific effects of CXCL10 that might either protect neurons or exacerbate damage.

These investigations extend CXCL10's known functions well beyond simple immune cell recruitment, positioning it as a multifunctional mediator in neurological injury and repair processes .

What are the current perspectives on targeting the CXCL10/CXCR3 axis in inflammatory and autoimmune diseases?

Current perspectives on targeting the CXCL10/CXCR3 axis in inflammatory and autoimmune diseases show promising therapeutic potential. Research has demonstrated that CXCL10 plays pathogenic roles in various inflammatory conditions, including experimental autoimmune prostatitis, by enhancing macrophage chemotaxis and proinflammatory cytokine secretion through activation of Erk and p38-MAPK signaling pathways.

The therapeutic approaches being explored include:

• Small molecule CXCR3 antagonists: Compounds like AMG487 have shown efficacy in preclinical models by blocking the interaction between CXCL10 and its receptor.

• Biologics targeting CXCL10: Monoclonal antibodies against CXCL10 are being evaluated for their ability to neutralize the chemokine in circulation.

• Gene silencing approaches: AAV-delivered shRNA targeting CXCL10 has demonstrated effectiveness in reducing disease severity in experimental models.

• Combination therapies: Targeting the CXCL10/CXCR3 axis alongside other inflammatory pathways may provide synergistic benefits in complex autoimmune conditions.

Challenges in this field include achieving tissue-specific targeting to avoid compromising beneficial immune responses and developing biomarkers to identify patients most likely to benefit from CXCL10/CXCR3-targeted therapies .

How might CXCL10 function as part of the broader chemokine network in coordinating immune responses?

CXCL10 functions as part of an intricate chemokine network that orchestrates immune responses through coordinated actions with other chemokines and cytokines. Current research perspectives on this network functionality include:

• Temporal coordination: CXCL10, induced by IFN-γ, typically acts during later phases of immune responses after initial inflammatory signals have established a pro-inflammatory environment. This temporal sequencing allows for progressive recruitment of different immune cell populations.

• Cell-specific targeting: While CXCL10 primarily attracts CXCR3-expressing T cells and NK cells, its coordination with other chemokines enables precise targeting of specific immune cell subsets to inflammatory sites.

• Amplification loops: CXCL10 secretion by monocytes, endothelial cells, and fibroblasts in response to IFN-γ creates positive feedback loops, as recruited T cells produce more IFN-γ, further amplifying the response.

• Antagonistic relationships: CXCL10 can function antagonistically with other chemokines that promote angiogenesis, providing balanced regulation of tissue remodeling during inflammation.

• Cross-regulation with cytokine networks: CXCL10 interacts with broader cytokine networks, influencing not just cell recruitment but also activation states, polarization, and effector functions of immune cells.

This network perspective is crucial for understanding the context-dependent effects of CXCL10 and for developing more sophisticated therapeutic approaches that modulate specific aspects of immune coordination rather than blocking single mediators .