IL12 Mouse, Sf9

What is the molecular structure of IL12 Mouse, Sf9 recombinant protein?

IL12 Mouse recombinant produced in the baculovirus expression system is a glycosylated disulfide-linked heterodimer comprised of two subunits: IL12A (p35) and IL12B (p40). The IL12A subunit spans amino acids 23-215 (199 aa total) with a molecular weight of 22.5 kDa, while IL12B spans amino acids 23-335 (319 aa total) with a molecular weight of 35.7 kDa. The total predicted molecular mass is 58.3 kDa, though it typically appears higher on SDS-PAGE due to glycosylation. The IL12A subunit includes a C-terminal 6-amino acid His-Tag for purification purposes .

What are the optimal storage conditions for IL12 Mouse, Sf9?

For optimal stability and activity retention, IL12 Mouse, Sf9 should be stored at -20°C for long-term storage. The protein is typically provided in a solution containing phosphate-buffered saline (pH 7.4) with 10% glycerol at a concentration of 0.5 mg/ml. For short-term usage (2-4 weeks), the protein can be stored at 4°C if the entire vial will be used within that period. Critically, freeze-thaw cycles should be avoided as they can compromise protein integrity and biological activity .

How is the purity and activity of IL12 Mouse, Sf9 typically assessed?

The purity of IL12 Mouse, Sf9 is generally greater than 95.0% as determined by SDS-PAGE analysis. For biological activity assessment, the standard method involves measuring IFN-γ production using an ELISA assay with NK-92 human natural killer cells. The typical ED50 (effective dose for 50% maximal response) for this effect is less than or equal to 10 ng/ml, which serves as a benchmark for functional quality control .

How should IL12 Mouse, Sf9 be implemented in immune cell activation studies?

When designing immune cell activation experiments, IL12 Mouse, Sf9 can be used to stimulate T cell and NK cell responses. A methodological approach involves culturing murine splenocytes or purified NK cells at 1-2 × 10^6 cells/ml in complete medium supplemented with IL12 at concentrations ranging from 1-50 ng/ml. For optimal results, culture for 24-72 hours before assessing activation markers (CD69, CD25), cytokine production (particularly IFN-γ), or cytotoxic activity against target cells. Include appropriate controls such as unstimulated cells and cells stimulated with control proteins to establish baseline responses .

What experimental models are suitable for studying IL12-mediated anti-tumor immunity?

Humanized mouse models offer a powerful system for studying IL12-mediated anti-tumor immunity. A validated approach involves using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice reconstituted with human CD34+ hematopoietic stem cells. Following immune reconstitution (approximately 12 weeks), engraft human tumor cells subcutaneously (e.g., rhabdomyosarcoma A204 cells). For IL12 treatment, administer tumor-targeted IL12 (2-5 μg/mouse) alone or in combination with IL-2 (1-2 μg/mouse) or IL-7 via intratumoral or perilesional injections every 2-3 days. Monitor tumor growth, survival, and analyze tumor-infiltrating lymphocytes for cytokine production and cytotoxic activity to assess treatment efficacy .

How can IL12 Mouse, Sf9 be used to study Th1 cell differentiation?

To study Th1 cell differentiation using IL12 Mouse, Sf9, isolate naive CD4+ T cells from mouse spleens using magnetic bead separation. Culture cells (1 × 10^6 cells/ml) in complete medium with plate-bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies. Add IL12 Mouse, Sf9 at 10 ng/ml to promote Th1 differentiation. For comparison, establish parallel cultures with IL-4 (10 ng/ml) to promote Th2 differentiation. After 3-5 days, analyze cells for characteristic Th1 markers (T-bet, IFN-γ) by flow cytometry, qPCR, and ELISA. This approach allows for direct assessment of IL12's role in T helper cell lineage commitment .

What is the relationship between IL12 and IFN-γ production in immune responses?

IL12 serves as a critical upstream regulator of IFN-γ production in the immune response cascade. Experimental evidence shows that IL12 knockout mice exhibit severely reduced parasite-antigen-specific IFN-γ responses (approximately 6 ng/ml on day 7 and 1.5 ng/ml on day 14 of infection) compared to wild-type mice following Trypanosoma cruzi infection. Notably, this reduction occurs without significant increases in IL-4 or IL-10 production, indicating that IL12 specifically drives Th1-type responses rather than suppressing Th2 responses. In experimental systems, treating spleen cell cultures with high concentrations (100 μg) of anti-IL12 neutralizing antibodies reduces IFN-γ production by 80-90%, confirming the direct dependency of IFN-γ production on IL12 stimulation .

How does IL12 interact with other cytokines in regulating immune responses?

IL12 functions within a complex cytokine network where its activity is modulated by and modulates other cytokines. Research demonstrates that in IL12 knockout mice, residual IFN-γ production is down-regulated by IL-10 but not by IL-4. Additionally, TNF-α can stimulate IFN-γ synthesis in both wild-type and IL12 knockout mice, suggesting a compensatory mechanism. When using IL12 Mouse, Sf9 in experimental settings, consider co-administration with IL-2 or IL-7, as these combinations have shown enhanced anti-tumor effects including enhanced cytotoxic T and NK cell responses, tumor regression, and 100% host survival in humanized mouse models. For optimal experimental design, include cytokine blocking antibodies to delineate the specific contributions of each cytokine in the observed biological effects .

What mechanisms underlie IL12-mediated enhancement of cytotoxic immune responses?

IL12 enhances cytotoxic immune responses through multiple coordinated mechanisms. When applied experimentally, IL12 Mouse, Sf9 stimulates the proliferation of activated T cells and NK cells while simultaneously upregulating the expression of cytotoxic mediators (perforin, granzymes) and death receptor ligands (FasL). Additionally, IL12 promotes IFN-γ production, which further activates macrophages to produce nitric oxide and other antimicrobial factors. In tumor models, IL12 combined with IL-2 enhances infiltration of tumors by cytotoxic T cells and NK cells, increases expression of adhesion molecules on tumor vasculature, and reduces immunosuppressive factors in the tumor microenvironment. For experimental assessment of these mechanisms, analyze cytotoxic activity using target cell lysis assays, flow cytometric detection of cytotoxic mediators, and measurement of nitric oxide production by the Griess reaction .

How can attenuated IL12 fusion proteins be designed for targeted tumor therapy?

Advanced research with IL12 Mouse, Sf9 includes developing attenuated forms with tumor-specific activation. A methodological approach involves constructing fusion proteins consisting of IL12 joined to a specific inhibitor (such as a single-chain fragment variable [scFv] antibody) via a protease cleavage sequence (cs). The inhibitor binds to IL12, attenuating its activity until cleaved by tumor-associated proteases like matrix metalloproteinases (MMPs).

To develop such constructs:

• Identify scFv that bind to independent epitopes on IL12 using phage display or hybridoma technology

• Incorporate these scFv into IL12 fusion proteins containing either an MMP cleavage sequence or a scrambled control sequence

• Verify attenuation of the intact fusion protein using functional assays (proliferation of CTLL-2 cells and IFN-γ induction)

• Confirm restoration of IL12 activity upon cleavage by the target protease (e.g., MMP9)

This approach has demonstrated successful attenuation of IL12 activity in the intact fusion protein and increased biological activity after MMP9 cleavage, potentially reducing unwanted side effects of systemic IL12 delivery .

What are the critical factors in designing IL12-based immunotherapies for infectious disease models?

When designing IL12-based immunotherapies for infectious disease models using IL12 Mouse, Sf9, several critical factors must be considered. Studies with Trypanosoma cruzi infection models demonstrate that IL12 knockout mice become highly susceptible to infection, characterized by increased parasitemia and mortality. This susceptibility is associated with severe depletion of parasite-antigen-specific IFN-γ responses and reduced nitric oxide production.

For effective experimental design:

• Determine optimal timing of IL12 administration relative to infection (prophylactic vs. therapeutic)

• Establish appropriate dosing regimens (typically 0.5-2 μg/day for murine models)

• Consider combination with other immune modulators (e.g., anti-IL-10 antibodies) to enhance efficacy

• Include comprehensive immune monitoring (cytokine profiles, cellular phenotyping, pathogen burden)

• Evaluate both innate and adaptive immune parameters, as IL12 affects both arms of immunity

Notably, in knockout models, the residual IFN-γ production is regulated differently than in wild-type mice, with IL-10 playing a significant down-regulatory role, suggesting potential benefit from combined IL12 administration with IL-10 blockade .

How can systems biology approaches be applied to understand IL12 signaling networks?

Systems biology approaches offer powerful tools to comprehensively map IL12 signaling networks using IL12 Mouse, Sf9 as a stimulus. A methodological framework includes:

• Phosphoproteomic analysis: Stimulate relevant immune cells (NK cells, T cells) with IL12 Mouse, Sf9 (10-50 ng/ml) for various time points (5, 15, 30, 60 min) and perform mass spectrometry-based phosphoproteomics to identify activated signaling nodes.

• Transcriptomic profiling: Conduct RNA-seq on IL12-stimulated cells at multiple time points (2, 6, 12, 24 hours) to identify gene expression programs initiated by IL12 signaling.

• Network analysis: Apply computational algorithms to integrate phosphoproteomic and transcriptomic data, constructing temporal signaling networks that link IL12 receptor engagement to functional outcomes.

• Perturbation studies: Systematically inhibit key nodes in the identified networks using small molecule inhibitors or genetic approaches (CRISPR-Cas9) to validate the importance of specific pathways.

• Mathematical modeling: Develop predictive models of IL12 signaling that can forecast cellular responses to various stimulation conditions or combination therapies.

This systems approach has revealed that IL12 signaling extends beyond the canonical JAK-STAT pathway to include cross-talk with multiple signaling cascades, including MAPK and PI3K pathways, providing a more comprehensive understanding of IL12 biology .

What strategies can address variability in IL12 bioactivity across experimental systems?

Variability in IL12 Mouse, Sf9 bioactivity across different experimental systems can significantly impact research outcomes. To address this challenge:

• Standardize activity units: Always perform a bioactivity assay with each new lot of IL12 using a standard readout such as IFN-γ ELISA with NK-92 cells. Define working concentrations in terms of biological activity units rather than absolute protein concentration.

• Include internal standards: In each experiment, include a reference control (e.g., commercial recombinant IL12 with established activity) to normalize responses across experiments.

• Validate cell responsiveness: Before experiments, confirm that target cells express functional IL12 receptors using flow cytometry or response to standard IL12 stimulation.

• Account for species specificity: Remember that mouse IL12 has species-specific activity and may not fully activate human cells, which is particularly important in humanized mouse models.

• Optimize storage and handling: Minimize freeze-thaw cycles and prepare single-use aliquots of IL12 to maintain consistent activity throughout a series of experiments.

These methodological considerations are essential for generating reproducible results when working with IL12 Mouse, Sf9 across different experimental platforms .

How can researchers optimize IL12 Mouse, Sf9 use in complex in vivo models?

Optimizing IL12 Mouse, Sf9 use in complex in vivo models requires careful consideration of several methodological aspects:

• Delivery method selection: Choose between systemic (intravenous, intraperitoneal) or local (intratumoral, site of infection) administration based on experimental goals. Local delivery typically requires lower doses and minimizes systemic toxicity.

• Dosing optimization: Conduct dose-response studies (typically starting with 0.1-1 μg/mouse) with close monitoring for signs of toxicity such as weight loss, splenomegaly, or elevated liver enzymes.

• Timing considerations: For tumor models, establish optimal treatment schedules (e.g., every 2-3 days) and determine whether treatment should begin when tumors are small or after they've reached a certain size.

• Combination strategies: When combining IL12 with other immunomodulators like IL-2, determine whether sequential or simultaneous administration yields superior results.

• Pharmacokinetic monitoring: Consider tagging IL12 with a reporter or using ELISA to track IL12 levels in serum and tissues to understand biodistribution and clearance rates.

These approaches have been validated in humanized mouse models, where tumor-targeted IL12 coupled with IL-2 showed enhanced cytotoxic T and NK cell responses, tumor regression, and 100% host survival .