I TAC Mouse

What is I-TAC/CXCL11 and what are its primary functions in mice?

I-TAC (Interferon-inducible T-cell alpha chemoattractant), also known as CXCL11, is a member of the CXC chemokine family with a molecular weight of 8-11 kDa. In mice, CXCL11 functions primarily as a chemoattractant for activated T cells through interaction with the CXCR3 receptor. The mouse CXCL11 is a 9.0 kDa protein containing 79 amino acid residues in its mature form, including four highly conserved cysteine residues characteristic of CXC chemokines. This chemokine plays crucial roles in regulating calcium release and inducing chemotactic responses in activated T-cells. Mouse CXCL11 cDNA encodes a 100 amino acid residue precursor protein with a cleavable signal sequence .

How does mouse I-TAC/CXCL11 compare structurally and functionally to human I-TAC/CXCL11?

Mouse CXCL11 shares approximately 68% amino acid sequence homology with human CXCL11. The human version contains 73 amino acid residues in its mature form, compared to 79 residues in the mouse version. Despite these differences, both species' versions interact with the CXCR3 receptor, though potentially with different binding affinities. Mouse CXCL11, like its human counterpart, is actively involved in immune cell chemotaxis, particularly for activated T cells. The structural differences between species should be considered when designing experiments that aim to translate findings from mouse models to human applications .

What is the relationship between I-TAC/CXCL11 and other chemokines in the mouse immune system?

I-TAC/CXCL11, together with MIG (CXCL9) and IP-10 (CXCL10), constitutes a subset of chemokines that function as ligands for the CXCR3 receptor. Among these three chemokines, I-TAC/CXCL11 demonstrates the highest binding affinity for CXCR3. This receptor is primarily expressed on activated Th1 cells and NK cells, indicating the importance of these chemokines in Th1-type immune responses. Interestingly, these three chemokines also function as antagonists for CCR3, a chemokine receptor preferentially expressed on activated Th2 cells, suggesting a regulatory role in balancing Th1 versus Th2 immune responses .

How can I effectively detect and quantify I-TAC/CXCL11 in mouse experimental samples?

Multiple methods exist for reliable detection and quantification of I-TAC/CXCL11 in mouse samples:

For protein quantification, sandwich ELISA is widely recommended. The Mouse I-TAC/CXCL11 ELISA employs a target-specific antibody pre-coated in microplate wells to capture I-TAC/CXCL11 from samples. The addition of a detector antibody followed by substrate solution generates a measurable signal proportional to I-TAC/CXCL11 concentration. This method can quantitate I-TAC/CXCL11 in mouse serum, plasma, or cell culture medium and recognizes both natural and recombinant forms .

For confirmation of antibody specificity or neutralization capacity, functional assays can be performed. For example, chemotaxis assays using the BaF3 mouse pro-B cell line transfected with human CXCR3 demonstrate dose-dependent migration in response to recombinant Mouse CXCL11/I-TAC. The chemotactic response can be neutralized by adding increasing concentrations of anti-Mouse CXCL11/I-TAC antibody, with a typical neutralization dose (ND50) of 2-12 μg/mL in the presence of 200 ng/mL recombinant Mouse CXCL11/I-TAC .

What experimental models are most appropriate for studying I-TAC/CXCL11 function in mice?

The choice of experimental model depends on the specific research question:

For analyzing basic expression patterns and regulation, wild-type mice with various inflammatory stimuli (particularly IFN-gamma) can be used to induce I-TAC/CXCL11 expression. IFN-gamma is a potent inducer of I-TAC/CXCL11 transcription, making IFN-gamma challenge models valuable for studying regulatory mechanisms .

For investigating I-TAC/CXCL11's role in disease processes, various disease models can be employed, including:

• Autoimmune disease models (multiple sclerosis, rheumatoid arthritis)

• Infectious disease models

• Cancer models, particularly those involving T cell recruitment

Cell culture systems using transfected cell lines, such as BaF3 mouse pro-B cells expressing human CXCR3, provide controlled environments for studying receptor-ligand interactions and downstream signaling pathways .

What are the critical considerations when designing chemotaxis assays to evaluate I-TAC/CXCL11 function?

When designing chemotaxis assays to study I-TAC/CXCL11 function, researchers should consider:

• Cell type selection: Use cells that naturally express or are transfected with CXCR3, such as activated T cells or the BaF3 mouse pro-B cell line transfected with human CXCR3.

• Dose-response relationship: Establish a complete dose-response curve for I-TAC/CXCL11, as optimal concentrations may vary depending on the cell type and experimental conditions.

• Specificity controls: Include neutralizing antibodies (such as Goat Anti-Mouse CXCL11/I-TAC Antibody) to confirm that observed chemotactic effects are specifically mediated by I-TAC/CXCL11.

• Quantification method: Select appropriate methods for quantifying cell migration, such as the Resazurin assay, which can measure the amount of cells that migrate through to the lower chemotaxis chamber.

• Comparison with other CXCR3 ligands: Compare I-TAC/CXCL11-induced chemotaxis with that induced by other CXCR3 ligands (CXCL9, CXCL10) to assess relative potency and potential functional differences .

What are the optimal storage and handling conditions for mouse I-TAC/CXCL11 reagents?

Proper storage and handling of mouse I-TAC/CXCL11 reagents are critical for maintaining their stability and functionality:

For antibodies and recombinant proteins:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain protein integrity

• Store unopened reagents at -20 to -70°C for up to 12 months from the date of receipt

• After reconstitution, store at 2 to 8°C under sterile conditions for up to 1 month for short-term use

• For extended storage after reconstitution, keep at -20 to -70°C under sterile conditions for up to 6 months

For recombinant proteins specifically, refer to the lot-specific Certificate of Analysis for detailed storage, handling, and reconstitution information. When working with Mouse CXCL11/I-TAC recombinant protein, reconstitution should be performed carefully according to manufacturer instructions to ensure proper protein folding and activity .

How can I troubleshoot inconsistent results in I-TAC/CXCL11 detection assays?

When encountering inconsistent results in I-TAC/CXCL11 detection assays, consider the following troubleshooting approaches:

• Sample preparation issues:

  • Ensure proper sample collection and processing to prevent protein degradation

  • Verify that sample storage conditions maintain protein integrity

  • Check for potential interfering substances in complex biological samples

• Assay-specific considerations:

  • For ELISA: Validate antibody specificity, optimize antibody concentrations, ensure proper blocking and washing steps

  • For functional assays: Confirm cell viability and receptor expression, optimize assay conditions

• Reagent quality control:

  • Verify the quality and activity of recombinant proteins

  • Confirm antibody specificity using positive and negative controls

  • Consider lot-to-lot variations in reagents

• Validation with multiple methods:

  • Confirm findings using complementary techniques (e.g., ELISA, Western blot, functional assays)

  • Include appropriate controls in each experiment .

What factors affect the stability and biological activity of recombinant mouse I-TAC/CXCL11?

Several factors can impact the stability and biological activity of recombinant mouse I-TAC/CXCL11:

• Production system: The expression system used for producing recombinant proteins can affect post-translational modifications and folding. For instance, E. coli-derived recombinant mouse CXCL11/I-TAC (spanning Phe22-Met100) may have different properties compared to mammalian cell-expressed versions .

• Storage conditions: Temperature fluctuations and repeated freeze-thaw cycles can lead to protein denaturation and loss of activity. Storage at -20 to -70°C is generally recommended for long-term preservation .

• Buffer composition: The reconstitution buffer's pH, ionic strength, and presence of stabilizing agents can significantly impact protein stability.

• Protein concentration: Very dilute solutions may lead to protein adsorption to container surfaces, while highly concentrated solutions might promote aggregation.

• Presence of proteases: Contaminating proteases in biological samples can degrade I-TAC/CXCL11, reducing its detectability and activity.

For maximum retention of biological activity, recombinant Mouse CXCL11/I-TAC should be handled according to manufacturer guidelines, with particular attention to recommended reconstitution procedures and storage conditions .

How should I design experiments to investigate the regulatory mechanisms of I-TAC/CXCL11 expression?

When investigating regulatory mechanisms of I-TAC/CXCL11 expression in mice, consider these experimental design principles:

• Stimulation protocols:

  • IFN-gamma is a potent inducer of I-TAC/CXCL11 transcription and should be included as a positive control

  • Compare effects of different cytokines and inflammatory mediators to identify specific regulatory pathways

  • Include time-course experiments to capture both early and late regulatory events

• Cell/tissue selection:

  • Target tissues known to express I-TAC/CXCL11, including peripheral blood leukocytes, pancreas, liver, thymus, spleen, lung, small intestine, placenta, and prostate

  • Include primary cells and relevant cell lines for in vitro studies

• Molecular approaches:

  • Analyze both transcriptional (mRNA) and translational (protein) regulation

  • Consider the role of the gene's structure, including its 4 exons and multiple polyadenylation signals that might reflect cell-specific regulation

• Signaling pathway analysis:

  • Investigate the role of JAK-STAT pathways, particularly STAT1, which is activated by IFN-gamma

  • Use specific inhibitors to dissect the contribution of different signaling pathways

• Genetic approaches:

  • Compare wild-type and gene-modified mice (e.g., IFN-gamma knockout) to establish cause-effect relationships .

What controls are essential when studying specificity of I-TAC/CXCL11 interactions with CXCR3?

When investigating the specificity of I-TAC/CXCL11 interactions with CXCR3, include these essential controls:

• Receptor specificity controls:

  • Compare responses in CXCR3-positive versus CXCR3-negative cells

  • Use CXCR3 antagonists to confirm receptor involvement

  • Include cells expressing related chemokine receptors to assess cross-reactivity

• Ligand specificity controls:

  • Include other CXCR3 ligands (CXCL9, CXCL10) to compare relative potency and efficacy

  • Use neutralizing antibodies specific for I-TAC/CXCL11 to confirm the observed effects are attributable to this chemokine

  • Test structurally modified or truncated versions of I-TAC/CXCL11 to identify critical binding domains

• Functional validation:

  • Demonstrate dose-dependent responses (e.g., chemotaxis of BaF3 cells expressing CXCR3)

  • Confirm that neutralizing antibodies inhibit the functional response (e.g., the ND50 for anti-Mouse CXCL11/I-TAC antibody is typically 2-12 μg/mL in chemotaxis assays)

• Signaling pathway verification:

  • Monitor receptor-mediated signaling events (e.g., calcium flux, ERK phosphorylation)

  • Confirm that these signaling events are inhibited by receptor antagonists or neutralizing antibodies .

What are the strengths and limitations of different detection methods for mouse I-TAC/CXCL11?

This comparative analysis highlights the importance of selecting appropriate detection methods based on the specific research question and available samples. For comprehensive studies, combining multiple detection methods is recommended to validate findings across different experimental approaches .

What are promising new technologies for studying I-TAC/CXCL11 biology in mouse models?

The field of chemokine research is rapidly evolving with several emerging technologies that offer new opportunities for investigating I-TAC/CXCL11 biology:

• Single-cell RNA sequencing enables comprehensive profiling of I-TAC/CXCL11 and CXCR3 expression at the single-cell level, revealing heterogeneity in expression patterns across different cell populations within tissues.

• CRISPR/Cas9 gene editing provides precise manipulation of the I-TAC/CXCL11 gene or its regulatory elements, facilitating the creation of novel mouse models with specific mutations or reporter constructs.

• Intravital microscopy allows real-time visualization of I-TAC/CXCL11-mediated cell migration in living tissues, providing insights into the dynamics of chemokine-directed cell movement in physiological contexts.

• Proteomics approaches can identify novel interaction partners and post-translational modifications of I-TAC/CXCL11, expanding our understanding of its regulatory network.

• Computational modeling integrates experimental data to predict how changes in I-TAC/CXCL11 expression or function might impact immune responses in complex disease settings.

These advanced technologies, when combined with traditional approaches, will significantly enhance our understanding of I-TAC/CXCL11 biology and its therapeutic potential in various disease contexts .