Recombinant Rat C-X-C motif chemokine 10 protein (Cxcl10) (Active)

What is the molecular structure and basic properties of Recombinant Rat CXCL10?

Recombinant Rat CXCL10 (also known as IP-10) is an 8.6 kDa protein comprising 77 amino acids. Its structure includes four conserved cysteine residues characteristic of the CXC chemokine superfamily. These conserved cysteines are crucial for maintaining the protein's functional three-dimensional conformation and biological activity . The protein was first cloned in 1985 and belongs to a family of highly inducible primary response genes. In research applications, understanding this structural foundation is essential for experimental design and interpretation of functional studies.

What are the main cellular sources of CXCL10 in experimental models?

CXCL10 is produced by multiple cell types depending on the experimental context. In murine models, Sertoli cells have been shown to produce CXCL10 in response to Mumps virus (MuV) infection . CD8+ T cells can also produce CXCL10 in response to specific stimulation, where it functions in a paracrine fashion to promote responses of other virus-specific CD8+ T cells . Additionally, research has demonstrated that testicular somatic cells including testicular macrophages, Sertoli cells, Leydig cells, and peritubular myoid cells can express CXCL10 in response to viral infections, whereas male germ cells typically do not produce this chemokine . This cell-specific expression pattern is important when designing experiments that aim to study CXCL10 production in different physiological and pathological contexts.

How does CXCL10 signal transduction work in experimental systems?

CXCL10 primarily signals through the CXCR3 receptor, which it shares with another chemokine called Mig . In experimental systems, CXCR3 is constitutively expressed on male germ cells, making them responsive to CXCL10 signaling . The downstream effects of CXCL10-CXCR3 interaction include activation of caspase-3 in certain contexts, such as germ cell apoptosis induced by MuV infection . In CD8+ T cell responses, CXCL10 signaling through CXCR3 promotes clonal expansion and facilitates the formation of CD8+ effector T cell pools in the lymph node microenvironment . When designing experiments to study CXCL10 signaling, researchers should consider using neutralizing antibodies against CXCR3 or caspase inhibitors as experimental controls to validate specificity of the observed effects.

What are the optimal conditions for reconstitution and storage of Recombinant Rat CXCL10?

For optimal reconstitution and storage of Recombinant Rat CXCL10, researchers should follow the lot-specific Certificate of Analysis provided with the product. Generally, the protein is shipped at ambient temperature but requires specific storage conditions to maintain activity .

A methodological approach includes:

• Reconstitution in sterile water or an appropriate buffer (often PBS with 0.1% BSA)

• Gentle agitation rather than vortexing to prevent protein denaturation

• Aliquoting the reconstituted protein to minimize freeze-thaw cycles

• Storage at -80°C for long-term preservation or -20°C for short-term storage

Researchers should validate protein activity after reconstitution using functional assays specific to their experimental design, such as chemotaxis assays or receptor binding studies.

How can researchers accurately measure CXCL10 levels in experimental samples?

Multiple methodological approaches can be employed to measure CXCL10 levels in experimental samples:

• Enzyme-linked immunosorbent assay (ELISA) is widely used for quantitative measurement of CXCL10 in cell culture supernatants, serum, or tissue lysates. Studies measuring CXCL10 in testicular lysates after MuV injection have successfully employed this method .

• For temporal changes in CXCL10 levels during treatment monitoring, standardization of sample means and calculation of fold-change values relative to previous collection time points can provide more accurate assessments of CXCL10 dynamics .

• When comparing slow versus fast responders in treatment scenarios (such as in tuberculosis studies), standardized mean difference (SMD) analysis within a random-effects model provides robust statistical evaluation .

• For tissue localization studies, immunohistochemistry or immunofluorescence co-staining approaches can identify both the source and target cells of CXCL10, as demonstrated in studies examining germ cell apoptosis in response to MuV infection .

Each method has specific advantages and limitations that should be considered based on the research question and available samples.

What controls should be included when studying CXCL10-induced cellular responses?

When studying CXCL10-induced cellular responses, several controls are essential for experimental rigor:

• Receptor blockade controls: Neutralizing antibodies against CXCR3 should be included to confirm specificity of observed effects. Studies have shown that anti-CXCR3 antibodies significantly inhibit CXCL10-induced male germ cell apoptosis .

• Pathway inhibition controls: Inhibitors of downstream signaling components, such as caspase-3 inhibitors in apoptosis studies, help validate the molecular mechanisms involved .

• Genetic controls: When available, cells or tissues from knockout models (CXCL10⁻/⁻ or CXCR3⁻/⁻) provide definitive evidence for CXCL10-specific effects. For example, MuV-induced germ cell apoptosis was significantly reduced in CXCL10⁻/⁻ mice compared to wild-type controls .

• Cytokine interdependence controls: Since CXCL10 expression can be induced by other cytokines like TNF-α, researchers should include conditions that block these upstream mediators. Pomalidomide, an inhibitor of TNF-α secretion, has been used to demonstrate the role of autocrine TNF-α in inducing CXCL10 expression .

• Time course controls: CXCL10 responses may vary temporally, so multiple time points should be assessed to capture the dynamics of the response.

How can CXCL10 be utilized as a biomarker in disease models and treatment monitoring?

CXCL10 has demonstrated significant potential as a biomarker in various disease models, particularly in treatment monitoring scenarios:

• Tuberculosis treatment monitoring: Meta-analysis has shown that reductions in CXCL10 levels during the first two months of anti-TB treatment correlate with positive treatment responses. Patients responding poorly to anti-TB treatment exhibited higher serum CXCL10 levels compared to those responding well (SMD: 1.23, 95% CI: -0.37–2.84) at the end of intensive treatment (2 months) .

• Viral infection models: In murine models of viral infection such as MuV, CXCL10 levels in testicular tissue correlate with germ cell apoptosis. Local injection of MuV into mouse testes significantly increases CXCL10 levels in testicular lysates after 24 hours, with subsequent increases in germ cell apoptosis at 48 hours post-infection .

• Immune response monitoring: During CD8+ T cell priming, CXCL10 levels can indicate the extent of T cell activation and clonal expansion. Genome-wide expression profiling has revealed the Cxcl10 gene as a target of CD27/CD70 costimulation in newly activated CD8+ T cells .

When implementing CXCL10 as a biomarker, researchers should:

• Establish baseline levels before intervention

• Collect samples at standardized time points

• Consider fold-changes relative to baseline rather than absolute values

• Account for potential confounding factors such as history of previous infection or treatment

What is the role of CXCL10 in T cell-mediated immune responses and how can it be experimentally manipulated?

CXCL10 plays several nuanced roles in T cell-mediated immunity that can be experimentally manipulated:

• CD8+ T cell priming: Rather than affecting survival or proliferation directly, CXCL10 acts as a chemoattractant for other activated CD8+ T cells. It signals in a paracrine fashion to promote the generation of CD8+ effector T cell pools in the antigen-draining lymph nodes .

• CXCR3-dependent clonal expansion: CD8+ T cells require expression of CXCR3 for clonal expansion in CD27/CD70-dependent peptide-immunization models. This requirement can be experimentally manipulated using CXCR3 knockout models or receptor antagonists .

• CD27/CD70 costimulation pathway: CXCL10 is a downstream target of CD27/CD70 costimulation in newly activated CD8+ T cells. While CD27/CD70 costimulation promotes activated T cell survival, CXCL10 specifically influences the recruitment and participation of other primed CD8+ T cells in the effector pool .

Experimental manipulation approaches include:

• Use of neutralizing antibodies against CXCL10 or CXCR3

• CXCL10 or CXCR3 knockout models

• Exogenous administration of recombinant CXCL10

• Manipulation of upstream regulators like TNF-α or IFN-γ

• Use of viral vectors for targeted expression of CXCL10 in specific tissues

How does CXCL10 interact with other cytokines and chemokines in complex inflammatory networks?

CXCL10 functions within intricate inflammatory networks, interacting with multiple cytokines and chemokines:

• TNF-α interaction: TNF-α significantly upregulates CXCL10 production in Sertoli cells after MuV infection. This has been demonstrated through knockout models, where CXCL10 levels were significantly reduced in TNF-α⁻/⁻ cells compared to wild-type cells after viral challenge .

• IFN-γ pathway: While not explicitly detailed in the search results, CXCL10 is also known as interferon-gamma induced protein 10, indicating its strong connection to IFN-γ signaling pathways .

• Chemokine receptor sharing: CXCL10 shares its receptor CXCR3 with other chemokines, notably Mig (CXCL9). This receptor sharing creates potential for synergistic or competitive effects between these chemokines .

• Complementary biomarker potential: Studies have shown that CXCL9, which shares the CXCR3 receptor with CXCL10, demonstrates similar patterns of decline during treatment of conditions like tuberculosis. This suggests these chemokines may function in parallel or complementary pathways within inflammatory networks .

Experimental approaches to study these interactions include:

• Multiplex cytokine analysis to simultaneously measure multiple inflammatory mediators

• Combined knockout or neutralization of multiple pathway components

• Sequential inhibition studies to establish hierarchy within inflammatory networks

• Systems biology approaches to model complex interactions

How should researchers address heterogeneity in CXCL10 experimental data?

Heterogeneity in CXCL10 experimental data is a common challenge that requires systematic approaches to address:

What are common technical challenges in working with Recombinant Rat CXCL10 and how can they be overcome?

Researchers working with Recombinant Rat CXCL10 commonly encounter several technical challenges:

• Protein stability issues: CXCL10 can lose activity during storage or repeated freeze-thaw cycles. To overcome this:

  • Aliquot reconstituted protein to minimize freeze-thaw cycles

  • Add carrier proteins (such as 0.1% BSA) to prevent adsorption to tubes

  • Monitor protein activity using functional assays before critical experiments

• Species-specific differences: Rat CXCL10 may show different potency or receptor affinities compared to human or mouse orthologs. Researchers should:

  • Use species-matched experimental systems when possible

  • Validate cross-species reactivity when using in heterologous systems

  • Consider species-specific differences when interpreting results

• Dose-response variability: Different biological responses may require different concentrations of CXCL10. Researchers should:

  • Perform comprehensive dose-response studies

  • Include positive controls with known bioactivity

  • Validate activity in each experimental system

• Complex biological matrices: Sample matrices (serum, tissue lysates) may contain inhibitors or proteases that affect CXCL10 detection. Approaches include:

  • Adding protease inhibitors to samples

  • Optimizing sample dilution to minimize matrix effects

  • Including spike-recovery experiments to validate detection in complex matrices

How should conflicting results in CXCL10 research be interpreted and resolved?

When faced with conflicting results in CXCL10 research, investigators should take a systematic approach to interpretation and resolution:

• Experimental context evaluation: Different experimental conditions may explain conflicting results. For example, CXCL10 production varies by cell type, with studies showing it is produced by testicular somatic cells but not germ cells in response to viral infection . Carefully evaluate:

  • Cell types and their activation state

  • Species differences

  • Timing of measurements

  • Experimental stimuli used

• Methodological differences assessment: Conflict may arise from different measurement approaches:

  • Different assay sensitivities (e.g., ELISA vs. multiplex)

  • Different antibody clones recognizing different epitopes

  • Different detection methods (mRNA vs. protein)

  • In vitro vs. in vivo models

• Biological complexity consideration: CXCL10 functions within complex networks:

  • Effects may be context-dependent

  • Redundancy with other chemokines (like CXCL9) may mask effects in some systems

  • Threshold effects may exist where concentration determines outcome

• Reconciliation strategies:

  • Direct comparative studies using standardized methods

  • Meta-analysis with clear inclusion/exclusion criteria

  • Stratification of data based on identified variables

  • Collaborative cross-validation between laboratories

• Statistical power assessment: Inconsistent results may reflect underpowered studies:

  • Calculate appropriate sample sizes based on expected effect sizes

  • Consider Bayesian approaches to integrate prior knowledge

  • Report effect sizes and confidence intervals rather than just p-values

What novel applications of Recombinant Rat CXCL10 are emerging in immunological research?

Emerging applications of Recombinant Rat CXCL10 in immunological research include:

• T cell-based immunotherapies: CXCL10's role in CD8+ T cell priming and effector pool generation positions it as a potential adjuvant for vaccines or immunotherapies. Research shows that CXCL10 produced by primed CD8+ T cells in response to CD27/CD70 costimulation signals to other primed CD8+ T cells in the lymph node microenvironment .

• Biomarker development for treatment monitoring: Beyond tuberculosis, where decreasing CXCL10 levels correlate with treatment efficacy, this approach could extend to monitoring response to therapies for other infectious or inflammatory conditions .

• Reproductive immunology applications: Given CXCL10's role in testicular immune responses and its production by Sertoli cells during viral infections, it represents a promising target for understanding and potentially treating virus-induced male infertility .

• Combination with other chemokines: Studies showing similar patterns between CXCL9 and CXCL10 suggest potential for developing multi-chemokine panels for more precise disease monitoring and characterization .

• Targeted delivery systems: Developing strategies to deliver CXCL10 to specific anatomical locations could enhance localized immune responses while minimizing systemic effects.

What are promising areas for methodological advancement in CXCL10 research?

Several methodological advances show promise for enhancing CXCL10 research:

• Single-cell analysis techniques: These could reveal heterogeneity in CXCL10 production and response at the individual cell level, providing insights into cellular subpopulations that drive specific immune responses.

• In vivo imaging of CXCL10 gradients: Development of fluorescent or bioluminescent CXCL10 reporters could allow visualization of chemokine gradients in living tissues, enhancing understanding of how CXCL10 directs cellular trafficking.

• Systems biology approaches: Integrating CXCL10 data with other cytokines and cellular markers through computational modeling could better characterize complex inflammatory networks.

• Standardized biomarker panels: Creating standardized panels that include CXCL10 alongside other inflammatory markers like CXCL9 could improve disease monitoring consistency across studies and clinical applications .

• CRISPR-based functional genomics: Using CRISPR-Cas9 technology to systematically modify CXCL10 signaling components could reveal new molecular mechanisms and therapeutic targets.

• Trend meta-analysis methodologies: Further refinement of approaches like those used in tuberculosis studies, where temporal data from multiple time points is analyzed using random intercept models, could improve detection of clinically relevant changes in CXCL10 levels .

How might understanding of CXCL10 biology contribute to therapeutic interventions?

Understanding CXCL10 biology has several potential therapeutic applications:

• Anti-viral therapies: Since CXCL10 is involved in virus-induced pathology such as MuV-induced germ cell apoptosis, targeting this pathway could potentially mitigate viral damage to reproductive tissues .

• Treatment response prediction: Baseline CXCL10 levels have shown potential as predictors of treatment outcomes in tuberculosis, with elevated levels associated with poorer responses. This could help stratify patients for more personalized therapeutic approaches .

• T cell-based immunotherapies: CXCL10's role in facilitating CD8+ T cell clonal expansion suggests it could be manipulated to enhance immune responses in vaccination or cancer immunotherapy contexts .

• Targeted anti-inflammatory approaches: In conditions where CXCL10 contributes to pathological inflammation, targeted inhibition of the CXCL10-CXCR3 axis could provide more selective anti-inflammatory effects than broader immunosuppressive approaches.

• Reproductive medicine applications: Given CXCL10's role in testicular immune responses and potential impact on male fertility during viral infections, therapeutic strategies targeting this pathway could help preserve fertility in affected patients .

• Combinatorial approaches: The interaction between CXCL10 and other cytokines like TNF-α suggests that combination therapies targeting multiple points in these inflammatory networks might provide synergistic therapeutic effects .