Recombinant Mouse C-C motif chemokine 24 protein (Ccl24) (Active)

What is Recombinant Mouse C-C motif chemokine 24 protein (Ccl24) and what are its key structural features?

Recombinant Mouse C-C motif chemokine 24 protein (Ccl24), also known as eosinophil chemotactic protein 2 or small-inducible cytokine A24, is a chemokine protein expressed in E. coli systems for research applications. The protein corresponds to amino acids 27-119 of the native sequence, has a molecular weight of approximately 10.3 kDa, and is typically produced without fusion tags to maintain native structure and function. The protein is characterized by >97% purity as determined by SDS-PAGE analysis and contains <1.0 EU/μg endotoxin as measured by LAL method, making it suitable for sensitive immunological experiments .

How should Recombinant Mouse Ccl24 be stored and handled to maintain activity?

For optimal stability and activity maintenance, Recombinant Mouse Ccl24 should be stored at -80°C for long-term storage or at -20°C for shorter periods. Repeated freeze-thaw cycles significantly reduce protein activity and should be avoided. Working aliquots should be prepared upon first thaw and stored separately. When reconstituting lyophilized protein, sterile techniques must be employed, using either sterile water or buffer according to the manufacturer's specifications. After reconstitution, the protein should be used promptly or stored in single-use aliquots to preserve activity .

What are the primary biological functions of Ccl24 in mouse models?

Ccl24 functions primarily as a chemoattractant for eosinophils in mouse models, playing a significant role in inflammatory processes, particularly in allergic airway inflammation. Research demonstrates that Ccl24 mediates eosinophil chemotaxis to the lung during allergic responses, contributing to eosinophilic airway inflammation. It operates downstream of CD163 signaling pathways, with studies showing that CD163-deficient mice exhibit increased CCL24 production when challenged with house dust mite (HDM) allergens, resulting in enhanced eosinophilic airway inflammation and mucous cell metaplasia (MCM) .

How can Recombinant Mouse Ccl24 be utilized in in vitro migration assays?

For in vitro migration assays, Recombinant Mouse Ccl24 should be prepared in chemotaxis buffer (RPMI-1640 with 0.5-1% BSA) at concentrations ranging from 1-100 ng/mL, with 10 ng/mL typically serving as an optimal starting point. Researchers should use a transwell system with 5-8 μm pore size depending on the cell type being studied (5 μm for eosinophils). The lower chamber should contain the chemokine while the upper chamber holds the cell suspension (1-5×10^5 cells/well). After incubation (2-4 hours at 37°C, 5% CO2), migrated cells can be quantified by flow cytometry or direct counting. Controls should include both negative (buffer alone) and positive (established chemoattractant) conditions to accurately assess specific Ccl24-mediated migration .

What are the recommended concentration ranges for different experimental setups using Recombinant Mouse Ccl24?

The appropriate concentration of Recombinant Mouse Ccl24 varies by experimental application:

Concentration optimization is essential, as effective doses may vary based on cell type, specific experimental conditions, and the sensitivity of detection methods employed .

How can Recombinant Mouse Ccl24 be used to study eosinophilic inflammation in airway models?

To study eosinophilic inflammation using Recombinant Mouse Ccl24 in airway models, researchers can employ intranasal administration of 1-5 μg Ccl24 in 30-50 μL PBS to anesthetized mice, typically delivered in divided doses between nostrils. Alternatively, Ccl24 can be delivered via intratracheal instillation for more direct lung exposure. Bronchoalveolar lavage fluid (BALF) should be collected 24-72 hours post-administration to quantify recruited eosinophils. For more complex models, Ccl24 can be used alongside allergen challenges (such as house dust mite extract) to study the potentiation of inflammatory responses. Control groups should include vehicle-only administration and, when investigating neutralization effects, isotype control antibodies alongside anti-Ccl24 antibodies .

How does CD163 regulate Ccl24 expression in response to allergen exposure?

CD163 functions as a negative regulator of Ccl24 expression during allergen exposure. In wild-type mice, CD163 binds to allergens such as Dermatophagoides pteronyssinus peptidase 1 (Der p1) in a calcium-dependent manner, limiting subsequent Ccl24 production. CD163-deficient (Cd163^-/-) mice exhibit significantly increased CCL24 levels in bronchoalveolar lavage fluid (BALF) and lung tissue following house dust mite (HDM) challenge compared to wild-type counterparts. This increased Ccl24 production occurs without alterations in Th1, Th2, or Th17 cytokine profiles, suggesting a direct regulatory pathway. When alveolar macrophages from Cd163^-/- mice are stimulated ex vivo with HDM, they secrete elevated levels of Ccl24 compared to wild-type cells, demonstrating that CD163 expression on macrophages specifically modulates allergen-induced Ccl24 secretion .

What experimental approaches can be used to study the Ccl24-dependent inflammatory pathway?

To investigate the Ccl24-dependent inflammatory pathway, researchers should employ a multi-faceted approach:

• Genetic models: Utilize Cd163^-/- mice alongside Ccl24^-/- mice or Ccl24 receptor-deficient models to dissect pathway components.

• Neutralization studies: Implement site-directed delivery of neutralizing anti-Ccl24 antibodies (typically 10-50 μg per mouse) via intranasal administration before allergen challenge.

• Ex vivo cell restimulation: Isolate alveolar macrophages from allergen-challenged mice and restimulate with allergen (10-50 μg/mL) for 24 hours to measure Ccl24 secretion.

• Adoptive transfer experiments: Transfer allergen-pulsed bone marrow-derived cells between wild-type and knockout mice to identify the cell populations responsible for Ccl24-mediated effects.

• Proteomic analysis: Employ calcium-dependent binding assays to identify interactions between allergen components (such as Der p1) and regulatory proteins like CD163.

These approaches should be complemented with comprehensive analysis of inflammatory parameters, including differential cell counts in BALF, histological assessment of tissue inflammation, and quantification of mucous cell metaplasia .

How can Recombinant Mouse Ccl24 be used in the development of therapeutic strategies for allergic airway disease?

Developing therapeutic strategies targeting the Ccl24 pathway requires a systematic approach. Researchers should first establish dose-response relationships by administering neutralizing anti-Ccl24 antibodies (5-50 μg per mouse) at different timepoints around allergen challenge, analyzing both preventive (pre-challenge) and therapeutic (post-challenge) potential. Combination approaches testing anti-Ccl24 antibodies alongside standard treatments (corticosteroids, bronchodilators) should assess potential synergistic effects. For translation-oriented research, humanized mouse models expressing human CCL24 receptors can evaluate human-specific antibodies or small molecule inhibitors. Target validation requires demonstrating that neutralizing Ccl24 reduces not only acute inflammation parameters but also chronic remodeling markers such as subepithelial fibrosis and airway hyperresponsiveness. Pharmacokinetic studies should determine antibody persistence in lung tissue, while toxicology evaluations must address potential immunosuppressive effects .

What are the methodological considerations for studying Ccl24 cross-talk with other inflammatory pathways?

Investigating Ccl24 cross-talk with other inflammatory pathways requires attention to multiple experimental factors. Researchers should design time-course experiments (6, 24, 48, 72 hours post-stimulation) to distinguish between primary Ccl24 effects and secondary cascade activation. Cell-specific responses should be evaluated using sorted cell populations (eosinophils, macrophages, epithelial cells) to identify differential pathway activation. Receptor antagonist studies employing specific blockers for CCR3 (Ccl24 receptor) alongside other chemokine receptors can isolate pathway-specific effects. Phospho-flow cytometry or Western blot analysis should target key signaling nodes (MAPK, JAK/STAT, NF-κB) to identify shared signaling mechanisms between Ccl24 and other inflammatory mediators. RNA-seq or proteomics of Ccl24-stimulated cells compared to cells stimulated with other chemokines (CCL11, CCL17) can reveal unique and overlapping gene expression signatures, while validation in complex models (precision-cut lung slices or organoids) can confirm pathway interactions in more physiologically relevant systems .

What are common technical challenges when working with Recombinant Mouse Ccl24 and how can they be addressed?

When working with Recombinant Mouse Ccl24, researchers frequently encounter several technical challenges:

• Protein aggregation: To prevent aggregation, avoid vigorous shaking during reconstitution, use low-protein-binding tubes, and include 0.1% BSA as a carrier protein in working solutions.

• Loss of activity over time: Prepare single-use aliquots immediately after reconstitution and avoid repeated freeze-thaw cycles.

• Inconsistent cellular responses: Pre-test each new lot of recombinant protein using a standardized migration assay with a positive control cell line; ensure target cells express appropriate receptors through flow cytometry verification.

• High background in control samples: Thoroughly wash cell preparations to remove endogenous chemokines and use chemotaxis buffers with minimal serum to reduce non-specific migration.

• Endotoxin contamination effects: For inflammation studies, ensure working with preparations containing <1.0 EU/μg endotoxin and include polymyxin B controls when appropriate.

• Receptor desensitization: When performing sequential stimulation experiments, include appropriate rest periods (4-6 hours) between stimulations to allow receptor recycling .

How can researchers validate the biological activity of Recombinant Mouse Ccl24 before use in complex experiments?

Validating Recombinant Mouse Ccl24 biological activity requires a systematic approach using complementary methods:

• Chemotaxis assay: The gold standard for activity validation uses freshly isolated mouse eosinophils or the mouse eosinophilic cell line (AML14.3D10) in a transwell migration system. Active Ccl24 should induce dose-dependent migration with a typical ED50 of 1-10 ng/mL.

• Calcium flux assay: Load receptor-expressing cells with calcium-sensitive dye (Fluo-4 AM) and measure fluorescence changes upon Ccl24 addition, comparing signal strength to a reference standard.

• Receptor binding assay: Perform competitive binding assays using radiolabeled or fluorescently labeled reference chemokine and unlabeled test Ccl24.

• Phosphorylation of downstream targets: Western blot analysis for phosphorylated ERK1/2 or Akt in response to Ccl24 stimulation provides functional validation.

• Receptor internalization: Flow cytometry measurement of CCR3 surface expression before and after Ccl24 treatment can confirm receptor engagement.

Researchers should generate a standard curve with each validation method using a characterized reference lot and set acceptance criteria (typically within 75-125% of reference activity) for new lots .

What emerging research areas could benefit from utilizing Recombinant Mouse Ccl24?

Several emerging research areas present significant opportunities for Recombinant Mouse Ccl24 applications:

• Single-cell analysis of Ccl24 responses: Integrating Ccl24 stimulation with single-cell RNA sequencing or CyTOF analysis would enable identification of cell-specific response signatures and rare responder populations within heterogeneous tissues.

• Microbiome-Ccl24 interactions: Investigating how gut or lung microbiome alterations affect Ccl24 production and responsiveness could reveal novel microbiome-dependent inflammatory mechanisms, particularly relevant in allergic disease models.

• Ccl24 in tissue repair and fibrosis: Beyond acute inflammation, exploring Ccl24's potential roles in tissue repair processes, fibroblast activation, and extracellular matrix remodeling could uncover new functions beyond classical chemotaxis.

• Neuro-immune interactions: Studying how neural signals modulate Ccl24 production by tissue-resident immune cells, and conversely, how Ccl24 affects neuronal function, represents an understudied area at the neuro-immune interface.

• Combination immunotherapies: Evaluating Ccl24 neutralization alongside emerging biologics targeting other inflammatory pathways could identify synergistic therapeutic combinations for refractory allergic conditions .

How might advanced protein engineering approaches enhance the utility of Recombinant Mouse Ccl24 for research applications?

Protein engineering approaches offer several avenues to enhance Recombinant Mouse Ccl24 utility:

• Receptor-specific variants: Engineered Ccl24 variants with modified receptor binding domains could provide tools to selectively activate specific signaling pathways downstream of CCR3, allowing dissection of pathway-specific biological effects.

• Fluorescent fusion proteins: Creating functional Ccl24-fluorescent protein fusions would enable real-time visualization of chemokine distribution and receptor binding in live cell imaging studies.

• Extended half-life variants: Modifications such as PEGylation or fusion to albumin-binding domains could generate long-acting Ccl24 variants for sustained in vivo studies, reducing dosing frequency.

• Conditional activity chemokines: Developing protease-activated or photo-activatable Ccl24 variants would allow precise spatial and temporal control of chemokine activity in complex tissue environments.

• Antagonistic variants: Engineering competitive antagonist versions of Ccl24 that bind receptors without activating signaling would provide alternative tools to neutralizing antibodies for pathway inhibition studies.

These engineering approaches would significantly expand the experimental toolkit available for investigating Ccl24 biology across diverse research contexts .