Recombinant Rhesus Macaque Interleukin-5 protein (IL5) (Active)

What is Recombinant Rhesus Macaque IL-5 and how does it compare structurally to human IL-5?

Recombinant Rhesus Macaque IL-5 is a disulfide-linked homodimeric, non-glycosylated polypeptide protein containing two 115-amino acid chains with a total molecular mass of approximately 26.1 kDa . It is produced in E. coli expression systems and is typically tag-free with purity levels exceeding 98% as determined by SDS-PAGE . The protein is derived from Macaca mulatta (Rhesus macaque) with the expression region spanning amino acids 20-134 .

While the search results don't explicitly state the sequence homology between rhesus and human IL-5, similar cytokines like IL-9 and IL-33 from rhesus macaques share 78% and 73% nucleotide similarity respectively with their human counterparts . This significant homology contributes to the value of rhesus macaque models, as they closely simulate human physiological and immunological responses .

What biological activities characterize functional Recombinant Rhesus Macaque IL-5?

The biological activity of Recombinant Rhesus Macaque IL-5 is typically assessed using human TF-1 cell proliferation assays, with active protein showing an ED50 of less than 5 ng/ml, corresponding to a specific activity of >2.0 × 10^5 IU/mg . This proliferative response serves as a key functional indicator of the protein's activity.

IL-5 functions predominantly as an eosinophil differentiation factor, as indicated by one of its synonyms . It also plays roles in T-cell responses, hence its alternative name "T-cell replacing factor" (TRF) . The biological functions of IL-5 in rhesus macaques parallel many of those observed in humans, making it valuable for translational immunology research.

How can Recombinant Rhesus Macaque IL-5 be utilized in immunological research models?

Recombinant Rhesus Macaque IL-5 serves as a critical reagent for studying eosinophil development, differentiation, and function in non-human primate models. Researchers can employ this cytokine in various experimental contexts:

• In vitro stimulation: To examine the effects on isolated rhesus macaque immune cells, particularly eosinophil progenitors and mature eosinophils

• Ex vivo analyses: To investigate the impact on tissue samples from rhesus macaques

• Mechanistic studies: To understand signaling pathways in comparison with human systems

For experimental readouts, flow cytometric analysis following immunofluorescence staining represents a standard approach for evaluating cellular responses to IL-5 stimulation . Additionally, multiplex Luminex assays can be employed to assess cytokine networks activated by IL-5 .

What experimental designs are optimal for studying IL-5 functions in rhesus macaque models?

When designing experiments to investigate IL-5 functions in rhesus macaque models, researchers should consider:

• Baseline expression profiling: Use RT-PCR and Sanger sequencing to characterize endogenous IL-5 expression patterns across different immune cell populations

• Functional testing: Implement ELISA and proliferation assays to evaluate dose-dependent effects of the recombinant protein

• Age and developmental considerations: Account for age-dependent variations in immune parameters, as immunological metrics differ across developmental stages in rhesus macaques aged 0-55 weeks

• Rearing conditions: Control for or specifically investigate the impact of different rearing methods, as mother-reared versus nursery-reared macaques may exhibit distinct immunological profiles

What are the recommended reconstitution and storage protocols for Recombinant Rhesus Macaque IL-5?

For optimal results with Recombinant Rhesus Macaque IL-5:

• Perform a quick spin of the lyophilized protein vial before opening

• Reconstitute in sterile water to a concentration of at least 100 μg/ml

• Further dilute as needed in appropriate aqueous buffers

• Avoid vortexing, which may damage the protein structure

Although complete storage information is not provided in the search results, lyophilized IL-5 is noted to be stable at room temperature for up to three weeks . For longer-term storage, standard practices for recombinant proteins would suggest storage at -20°C or -80°C, with aliquoting to avoid freeze-thaw cycles.

How should researchers validate the activity of Recombinant Rhesus Macaque IL-5 in experimental systems?

Validating the biological activity of Recombinant Rhesus Macaque IL-5 should involve multiple approaches:

• Cell proliferation assays: Using human TF-1 cells as described in the literature, where functional IL-5 demonstrates an ED50 below 5 ng/ml

• Dose-response experiments: Testing a range of concentrations to establish optimal working dilutions for specific experimental systems

• Positive controls: Including established IL-5 preparations with known activity

• Orthogonal validation: Confirming activity through multiple readouts (e.g., proliferation, receptor binding, downstream signaling)

To ensure reproducibility, researchers should perform activity validation with each new lot of recombinant protein and periodically during extended storage.

How does IL-5 expression in rhesus macaques vary between immune cell subsets and under different stimulation conditions?

While detailed IL-5 expression data across rhesus macaque immune cell populations is not explicitly described in the search results, insights can be drawn from related cytokine studies. Analogous interleukins like IL-9 show expression in both peripheral blood mononuclear cells (PBMCs) and CD4+ T cells, with increased production following stimulation .

To comprehensively characterize IL-5 expression:

• Cell isolation: Separate distinct immune cell populations (T cells, B cells, eosinophils, mast cells) from rhesus macaque blood or tissues

• Baseline expression: Perform quantitative RT-PCR to measure constitutive IL-5 mRNA levels

• Stimulation conditions: Test various activators (e.g., PMA/ionomycin, allergens, specific cytokines) to identify optimal induction conditions

• Intracellular staining: Employ flow cytometry with anti-IL-5 antibodies to identify IL-5-producing cells at the single-cell level

What are the comparative immunological responses to IL-5 in rhesus macaques versus humans?

Rhesus macaques represent valuable models for human immunology because they closely simulate human physiological and immunological responses . For IL-5 specifically, researchers should consider:

• Receptor homology: The degree of sequence similarity between rhesus and human IL-5 receptors affects signaling outcomes

• Downstream pathway conservation: Evaluate whether signaling cascades activated by IL-5 are conserved between species

• Cellular response thresholds: Determine if rhesus cells require different concentrations of IL-5 to achieve comparable biological effects

• Cross-reactivity: Test whether human and rhesus IL-5 can effectively stimulate cells from the other species

These comparative analyses enhance the translational value of findings from rhesus macaque models to human immunology and pathology.

What are common challenges in detecting endogenous IL-5 in rhesus macaque samples?

Researchers working with rhesus macaque samples may encounter several challenges when attempting to detect endogenous IL-5:

• Limited antibody cross-reactivity: Human-specific antibodies may show variable recognition of rhesus IL-5

• Low baseline expression: IL-5 is often produced at low levels in the absence of specific stimulation

• Sample processing effects: Cytokine degradation during sample collection and processing can reduce detection sensitivity

To address these challenges:

• Validate antibodies specifically with recombinant rhesus macaque IL-5

• Optimize stimulation protocols to enhance IL-5 production before measurement

• Employ sensitive detection methods like multiplex Luminex assays, which have been successfully used for cytokine detection in rhesus macaque samples

• Consider measuring IL-5 at both protein and mRNA levels for confirmation

How can experimental conditions be optimized for studying IL-5 effects on rhesus macaque primary cells?

When working with primary cells from rhesus macaques:

• Media composition: Standard media formulations may require supplementation with rhesus-specific growth factors

• Seeding density: Determine optimal cell concentrations for IL-5 responsiveness

• Timing: Establish appropriate time courses for IL-5 stimulation, as effects may be both immediate and delayed

• Combinatorial cytokine effects: Test IL-5 alone and in combination with other cytokines that may synergize or antagonize its effects

For example, peripheral blood mononuclear cell preparation protocols have been established for rhesus macaques, as mentioned in the BNT162b vaccine study , which could be adapted for IL-5 response studies.