IL 3 Dog

Interleukin-3 Canine Recombinant
Shipped with Ice Packs
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Description

Introduction

Canine Interleukin-3 (IL-3), also referred to as IL-3 Dog, is a hematopoietic growth factor critical for the survival, proliferation, and differentiation of multipotent hematopoietic stem cells into myeloid lineage cells, including basophils, mast cells, granulocytes, and monocytes . Produced by T-cells, mast cells, and eosinophils, IL-3 exhibits species-specific activity and plays a key role in immune regulation and hematopoiesis .

Hematopoietic Activity

IL-3 promotes the expansion of myeloid progenitors, including pre-basophil and mast cell progenitors (pre-BMPs). In vitro studies demonstrate:

  • 10.5-fold increase in basophils and 3.5-fold increase in mast cells from pre-BMPs after IL-3 treatment .

  • Synergy with GM-CSF for basophil expansion but not mast cell differentiation .

In Vivo Effects

  • IL-3 immune complexes (IL-3c) injected in mice expanded pre-BMPs by 13.7-fold within 3 days .

  • No significant impact on granulocyte-macrophage progenitors (GMPs) or pro-BMPs, highlighting lineage specificity .

Receptor Regulation

  • IL-3 upregulates Il3ra (IL-3 receptor α-chain) mRNA and protein expression on pre-BMPs, enhancing self-sustained proliferation .

  • Gata2 mRNA expression increases but is dispensable for IL-3 receptor upregulation .

Cell Culture and Differentiation

  • ED50: <0.2 ng/mL for TF-1 cell proliferation, demonstrating high bioactivity .

  • Used to generate myeloid cells for studies on allergies, inflammation, and immune responses .

Therapeutic Development

  • Potential role in cell therapy: Supports T/NK cell activation and proliferation in preclinical models .

  • Toxicology studies: IL-3 derivatives (e.g., LY3509754 analogs) have faced challenges due to adverse effects in animal models, emphasizing the need for structural optimization .

Diagnostic Tools

  • Serves as an ELISA standard and Western blot control due to high batch-to-batch consistency .

Product Specs

Introduction
Interleukin-3 (IL-3) is a cytokine with potent growth-promoting effects on a wide range of hematopoietic cells, influencing their proliferation, differentiation, and apoptosis. Beyond its role in the blood system, IL-3 exhibits neurotrophic activity and might be implicated in neurological disorders.
Description
Recombinant Canine Interleukin-3, produced in E. coli, is a single-chain, non-glycosylated polypeptide consisting of 120 amino acids. With a molecular weight of 14 kDa, this purified IL-3 is obtained through proprietary chromatographic techniques.
Physical Appearance
White, lyophilized powder, sterile-filtered.
Formulation
Lyophilized from a 1 mg/ml solution in 1X PBS at pH 7.4.
Solubility
For reconstitution, it's recommended to dissolve the lyophilized IL-3 in sterile 18 MΩ-cm H2O at a concentration of at least 100 µg/ml. This solution can then be further diluted in other aqueous solutions.
Stability
While lyophilized IL-3 remains stable at room temperature for up to 3 weeks, it should ideally be stored in a desiccated state below -18°C. After reconstitution, store IL-3 at 4°C for 2-7 days. For long-term storage, keep it below -18°C. Avoid repeated freeze-thaw cycles.
Purity
Exceeds 97.0%, as determined by: (a) High-Performance Liquid Chromatography (HPLC) analysis and (b) SDS-PAGE analysis.
Biological Activity
The ED50, assessed by dose-dependent stimulation of TF-1 cells, is less than 0.2 ng/ml. This corresponds to a Specific Activity of 5,000,000 IU/mg.
Synonyms
MCGF (Mast cell growth factor), Multi-CSF, HCGF, P-cell stimulation factor, IL-3, MGC79398, MGC79399.
Source
Escherichia Coli.
Amino Acid Sequence
RPFSTDLPKQ YFTMINEIME MLNKSPSPSE EPLDSNEKET LLEDTLLRPN LDVFLNASSK FHKNGLLIWN NLKEFLPLLP TPTPRGEPIS IMENNWGDFQ RKLKKYLEAL DNFLNFKNKP.

Q&A

What is the molecular structure of canine IL-3?

Canine IL-3 is a cytokine involved in immune system signaling. Molecular analysis reveals that canine IL-3 cDNA includes a single open reading frame of 432 nucleotides, which encodes a 143 amino acid polypeptide. This structure was identified through reverse transcription polymerase chain reaction (RT-PCR) using RNA harvested from canine peripheral blood mononuclear cells (PBMCs) . The structural characterization of canine IL-3 provides fundamental insights into its function within the immune system and its potential role in various physiological and pathological processes.

How does canine IL-3 compare with IL-3 from other species?

Comparative genomic analysis demonstrates variable sequence homology between canine IL-3 and its counterparts in other mammals. Specifically, canine IL-3 shares 44.7% homology with bovine (cow) IL-3, 42.4% with ovine (sheep) IL-3, 37% with human IL-3, and only 23.7% with rat IL-3 . This relatively low cross-species conservation contrasts with other canine cytokines like IL-6, which shows 60.4% identity with human IL-6 and 77.2% with feline IL-6 . The limited homology between canine and human IL-3 (37%) has significant implications for research methodology, particularly regarding the specificity of reagents and the direct translation of findings between species.

What are the recommended methods for measuring IL-3 in canine samples?

Several methodological approaches can be employed for measuring IL-3 in canine samples:

  • Enzyme-Linked Immunosorbent Assay (ELISA): Traditional method requiring canine-specific antibodies for IL-3 detection

  • Multiplex Cytokine Assays: Methods such as the Milliplex Canine Cytokine Panel allow simultaneous measurement of multiple cytokines, potentially including IL-3

  • RT-PCR for gene expression analysis: Used for quantifying IL-3 mRNA expression rather than protein levels, as employed in the original characterization of canine IL-3

  • Flow Cytometry-Based Bead Assays: Combining features of ELISA and flow cytometry for potentially greater sensitivity

Method selection should consider research objectives, required sensitivity, sample type, and whether concurrent measurement of other cytokines would provide valuable contextual data.

How should researchers handle samples with IL-3 values below detection limits?

The challenge of measurements falling below detection limits is common in cytokine research. Based on approaches used with other canine cytokines, researchers should consider:

  • Statistical approaches for left-censored data:

    • Some investigators use a value that is half of the limit of detection

    • Others use the full value of the limit of detection for statistical analysis

    • Alternative approach: comparing the number of detectable measurements in different cohorts

  • Reporting considerations:

    • Clearly state the percentage of samples below detection limits

    • Consider reporting detection frequencies rather than absolute values

    • Use appropriate non-parametric statistics when a significant proportion of samples fall below detection limits

  • Methodological solutions:

    • Sample concentration techniques to enhance detection

    • Use of higher-sensitivity assays when available

    • Ex vivo stimulation protocols to induce measurable cytokine production

A standardized approach would facilitate better comparison between studies, especially given the frequency with which cytokine samples fall below detection limits in both healthy and affected dogs .

What experimental design considerations are critical when studying IL-3 in canine disease models?

When designing experiments to investigate IL-3 in canine disease models, researchers should address:

  • Timing of sample collection:

    • Studies have shown that cytokine levels can differ significantly between acute (less than 48 hours) and chronic (more than 48 hours) presentations of disease

    • Serum GM-CSF, IL-2, IL-6, and IL-18 concentrations have demonstrated significant differences based on disease duration in canine babesiosis

  • Clinical stratification:

    • Stratify subjects based on disease severity, as cytokine levels like IL-6 and MCP-1 have shown statistical differences between survivors and non-survivors in certain conditions

    • Consider breed, age, and size variations, as demonstrated in studies showing differential responses to treatments based on dog size

  • Control selection:

    • Include appropriate healthy controls matched for relevant variables

    • Consider including disease controls to establish specificity of cytokine changes

  • Complementary measurements:

    • Correlate cytokine levels with clinical parameters (e.g., IL-6 inversely correlating with serum total triiodothyronine concentrations)

    • Consider measuring multiple cytokines to understand network effects

  • Sample handling standardization:

    • Implement consistent processing, storage, and freeze-thaw protocols

    • Document sample stability conditions for IL-3

How do cytokine profiles including IL-3 change in different canine infectious diseases?

While specific IL-3 data in canine infectious diseases is limited, patterns from other cytokines provide insights:

What is the potential role of IL-3 in canine allergic and inflammatory conditions?

IL-3 likely plays an important role in canine allergic and inflammatory conditions:

How might IL-3 be involved in canine immune response to omega-3 supplementation?

Recent research on omega-3 fatty acid supplementation in dogs provides context for understanding potential IL-3 involvement:

  • Modulatory effects on inflammation:

    • Supplementation with approximately 70 mg of EPA + DHA/kg/day improved the Omega-3 Index and reduced pain scores in small and medium-sized dogs

    • Omega-3 supplementation for 16 weeks doubled the Omega-3 Index (+135%), which was greater than seen in past studies

  • Size-dependent responses:

    • Pain scores were significantly reduced by 38% in small dogs and by 30% in medium dogs, with no reduction in large dogs

    • Quality of life scores improved in small dogs but not in larger dogs

    • These size-dependent effects suggest differential inflammatory response mechanisms that might involve IL-3

  • Cytokine correlation potential:

    • While the study did not directly measure IL-3, the significant inverse relationship between pain and quality of life scores suggests underlying changes in inflammatory mediators

    • Future studies could investigate whether IL-3 levels correlate with response to omega-3 supplementation

What are the challenges in developing canine-specific IL-3 assays?

Developing reliable assays for canine IL-3 faces several challenges:

  • Limited cross-reactivity:

    • The relatively low homology (37%) between canine and human IL-3 means human assays may not reliably detect canine IL-3

    • Species-specific reagents are essential for accurate measurement

  • Sensitivity requirements:

    • Cytokines often circulate at low concentrations, requiring highly sensitive detection methods

    • Studies have reported detection challenges with other canine cytokines like IL-10 and IL-4, which were detected in less than 28% and 3% of clinical samples, respectively

  • Standardization issues:

    • Lack of standardized reference materials for canine IL-3

    • Inconsistent approaches to handling samples with values below detection limits

    • Variable sample processing methods affecting cytokine stability

  • Validation requirements:

    • Need for comprehensive validation of assay performance characteristics

    • Establishment of reference ranges in different dog breeds and ages

How can multiplex cytokine analysis advance our understanding of IL-3 in canine immunology?

Multiplex cytokine analysis offers several advantages for IL-3 research:

  • Comprehensive immune profiling:

    • Simultaneous measurement of multiple cytokines provides context for IL-3 function

    • Enables identification of coordinated cytokine responses and regulatory networks

  • Efficiency benefits:

    • Reduces sample volume requirements, particularly important for serial sampling

    • More cost-effective than running multiple single-cytokine assays

  • Pattern recognition:

    • Facilitates identification of disease-specific cytokine signatures

    • Allows correlation analysis between IL-3 and other immune mediators

    • Enables novel approaches comparing cytokine groups rather than individual molecules

  • Clinical applications:

    • Potential for developing cytokine-based diagnostic and prognostic tools

    • Monitoring treatment responses through changes in cytokine profiles

What are emerging directions for IL-3 research in veterinary immunology?

Several promising research directions could advance our understanding of IL-3 in canine health and disease:

  • Genetic and epigenetic regulation:

    • Investigation of breed-specific variations in IL-3 expression and function

    • Analysis of regulatory elements controlling IL-3 production in different immune cell populations

  • Single-cell technologies:

    • Characterization of IL-3-producing and IL-3-responsive cells at single-cell resolution

    • Analysis of cell-specific signaling pathways activated by IL-3

  • Therapeutic applications:

    • Development of recombinant canine IL-3 for potential therapeutic use

    • Investigation of IL-3 pathway inhibition for allergic and inflammatory conditions

    • Combination approaches with established therapies like Cytopoint

  • Comparative immunology:

    • Cross-species analysis of IL-3 function to identify conserved and divergent mechanisms

    • Leveraging canine models for understanding IL-3 biology relevant to human health

  • Clinical biomarker development:

    • Evaluation of IL-3 as part of cytokine panels for diagnosis and prognosis

    • Identification of IL-3-related biomarkers predictive of treatment response

What are the recommended protocols for optimal IL-3 mRNA expression analysis in canine samples?

Based on the successful cloning of canine IL-3, optimal protocols should incorporate:

  • Sample collection and processing:

    • Immediate stabilization of RNA in collected samples

    • Isolation of peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation

    • RNA extraction using methods that preserve integrity of cytokine transcripts

  • RT-PCR optimization:

    • Design of primers specific to canine IL-3 sequence

    • Use of appropriate housekeeping genes for normalization (validated in canine samples)

    • Inclusion of no-template and no-RT controls

  • Protocol details:

    • For initial characterization of canine IL-3, researchers successfully used RT-PCR with RNA harvested from canine PBMCs

    • This approach yielded a single open reading frame of 432 nucleotides encoding a 143 amino acid polypeptide

  • Verification approaches:

    • Sequence verification of PCR products

    • Use of positive controls (stimulated canine cells known to express IL-3)

    • Correlation with protein-level measurements when possible

How should researchers interpret contradictory cytokine data in canine studies?

Contradictory findings are common in cytokine research. To address this challenge:

What statistical approaches are most appropriate for analyzing IL-3 data in canine research?

Based on approaches used in canine cytokine research:

  • Handling non-detectable values:

    • Some investigators use half the limit of detection value

    • Others use the full value of the limit of detection

    • Alternative approach: comparing the number of detectable measurements in different cohorts

  • Correlation analyses:

    • Nonparametric correlation analyses (e.g., Kendall's Tau) are appropriate for cytokine data

    • Studies have used this approach to assess relationships between cytokines and clinical outcomes

    • Example: significant inverse relationship between pain and quality of life scores (rτ = −0.60, p < 0.0001)

  • Group comparisons:

    • For normally distributed data: paired t-tests for before-after comparisons

    • For non-normally distributed data: Wilcoxon signed-rank test or Mann-Whitney U test

    • For multiple group comparisons: ANOVA with appropriate post-hoc tests or Kruskal-Wallis test

  • Sample size considerations:

    • Power calculations should account for expected high variability in cytokine data

    • Consider pilot studies to estimate effect sizes and variability

  • Multivariate approaches:

    • Principal component analysis for exploring patterns in complex cytokine data

    • Cluster analysis to identify disease-specific cytokine signatures

    • Machine learning algorithms for predictive modeling

Canine IL-3 Research Challenges and Solutions
Challenge
Limited homology with human IL-3 (37%)
Values below detection limits
Variable cytokine responses by dog size
Acute vs. chronic phase differences
Lack of standardized protocols

Product Science Overview

Structure and Composition

The recombinant canine IL-3 protein is a single non-glycosylated polypeptide chain containing 120 amino acids . The amino acid sequence of this protein is as follows:

RPFSTDLPKQ YFTMINEIME MLNKSPSPSE EPLDSNEKET LLEDTLLRPN LDVFLNASSK FHKNGLLIWN NLKEFLPLLP TPTPRGEPIS IMENNWGDFQ RKLKKYLEAL DNFLNFKNKP

This sequence corresponds to the IL-3 protein derived from E. coli .

Biological Activity

IL-3 is a potent growth-promoting cytokine capable of supporting the proliferation of a broad range of hematopoietic cell types . It is involved in various cell activities such as cell growth, differentiation, and apoptosis . The recombinant canine IL-3 protein is fully biologically active when compared to the standard. The ED50, as determined by a cell proliferation assay using human TF-1 cells, is less than 0.2 ng/ml, corresponding to a specific activity of greater than 5.0 x 10^6 IU/mg .

Function and Applications

IL-3 plays a significant role in hematopoiesis by controlling the production, differentiation, and function of two related white cell populations of the blood: granulocytes and monocytes-macrophages . This makes it a critical factor in the immune response and in the development of various therapeutic applications.

Storage and Stability

The lyophilized preparation of recombinant canine IL-3 protein is stable for 12 months from the date of receipt when stored at -20 to -70 degrees Celsius, preferably desiccated . Upon reconstitution, the preparation can be stored for one month at 2-8 degrees Celsius under sterile conditions and for three months at -20 to -70 degrees Celsius . It is recommended to use a manual defrost freezer and avoid repeated freeze-thaw cycles .

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