Recombinant Mouse Eotaxin protein (Ccl11) (Active)

What analytical methods confirm the identity and purity of recombinant mouse CCL11?

Recombinant mouse CCL11 can be verified through multiple complementary techniques. SDS-PAGE analysis is commonly employed to assess protein size and purity, while mass determination via ESI-TOF (Electrospray Ionization-Time of Flight) provides precise molecular weight confirmation. For instance, the predicted molecular weight of mouse CCL11 is approximately 8496.21 Da, which can be verified through ESI-TOF mass spectrometry . Additionally, High-Performance Liquid Chromatography (HPLC) is utilized to evaluate protein homogeneity and purity. For optimal results, researchers should implement all three techniques to ensure comprehensive quality assessment before experimental use .

How does mouse CCL11 induce chemotaxis in experimental models?

Mouse CCL11 demonstrates dose-dependent chemotactic activity primarily through CCR3 receptor engagement. In experimental models, recombinant mouse CCL11 effectively chemoattracts BaF3 mouse pro-B cell lines transfected with mouse CCR3 receptors . The chemotactic response can be quantified by measuring cell migration through a membrane barrier using detection methods such as Resazurin fluorescence. Typically, effective chemotaxis is observed at concentrations around 10 ng/mL, though researchers should perform dose-response experiments (1-100 ng/mL range) to determine optimal concentrations for their specific cell types . This chemotactic effect can be neutralized in a dose-dependent manner using specific antibodies, such as Goat Anti-Mouse CCL11/Eotaxin Antigen Affinity-purified Polyclonal Antibody, with neutralization typically occurring at concentrations of 0.1-0.5 μg/mL .

What is the tissue distribution pattern of CCL11 in mouse models?

Immunohistochemical analyses have demonstrated that CCL11 is expressed in various mouse tissues, with particularly notable expression in the colon. When performing immunohistochemistry, perfusion-fixed frozen sections typically yield optimal results. Using specific antibodies like Goat Anti-Mouse CCL11/Eotaxin Antigen Affinity-purified Polyclonal Antibody at a concentration of 15 μg/mL (incubated overnight at 4°C), CCL11 shows cytoplasmic localization within colon tissues . For visualization, appropriate detection systems such as HRP-DAB (horseradish peroxidase-diaminobenzidine) can be employed with hematoxylin counterstaining. This approach allows researchers to examine the spatial distribution of CCL11 within tissues, providing insights into its potential functional roles in different anatomical contexts .

How does CCL11 contribute to cellular senescence pathways?

Recent research has revealed that CCL11 plays a significant role in promoting cellular senescence. Using computational prediction tools like PseudoCell, researchers have identified that increased CCL11 stimulation induces a dose-dependent activation of several key senescence-related pathways . Specifically, high CCL11 concentrations activate cytochrome b alpha (CYBA) and beta (CYBB) chains, which are responsible for reactive oxygen species (ROS) formation. This pro-oxidative environment subsequently triggers DNA damage signaling pathways .

When human lung fibroblast cells (MRC-5) are exposed to rhCCL11 (500ng/10^5 cells/mL), significant increases in phosphorylated H2A Histone Family Member X (γH2AX) and tumor suppressor protein TP53 (p-p53) are observed after 2 hours of treatment. Specifically, both the percentage of positive cells and mean fluorescence intensity (MFI) of γH2AX and p-p53 increase substantially (p<0.0001 and p=0.005 for γH2AX; p<0.0001 and p=0.0352 for p-p53, respectively) . Interestingly, these DNA damage responses appear to be transient, as they return to baseline levels 24 hours post-treatment, suggesting activation of DNA repair mechanisms .

What methodologies are recommended for neutralizing CCL11 activity in experimental settings?

For effective neutralization of mouse CCL11 in experimental systems, researchers should employ specific antibodies with demonstrated neutralizing capacity. Goat Anti-Mouse CCL11/Eotaxin Antigen Affinity-purified Polyclonal Antibody has shown efficacy in neutralizing CCL11-induced chemotaxis . When implementing neutralization studies, a dose-titration approach is essential. Typically, effective neutralization of 10 ng/mL recombinant mouse CCL11 requires antibody concentrations between 0.1-0.5 μg/mL .

For optimal neutralization protocols:

• Pre-incubate the neutralizing antibody with recombinant CCL11 for 30-60 minutes at room temperature

• Apply the mixture to your experimental system

• Include appropriate controls (isotype-matched antibodies) to confirm specificity

• Quantify neutralization efficiency using functional readouts such as chemotaxis inhibition

For chemotaxis neutralization assays, researchers can measure cell migration inhibition using methods like Resazurin fluorescence detection and calculate the ND50 (neutralizing dose that inhibits 50% of CCL11 activity) .

What experimental considerations are important when investigating CCL11's role in inflammatory diseases?

When investigating CCL11's role in inflammatory conditions, researchers should consider several methodological aspects. First, appropriate cell models must be selected; human lung fibroblasts (MRC-5) and CCR3-transfected BaF3 mouse pro-B cells have proven effective for studying CCL11-mediated effects . Flow cytometry represents an essential technique for quantifying cellular responses, using a 3-laser configuration (Violet 405 nm, Blue 488 nm, and Red 633 nm) for optimal detection .

For secretion studies, ELISA (enzyme-linked immunosorbent assay) methods are recommended. Researchers should seed cells at approximately 1×10^5 cells/well and treat with appropriate CCL11 concentrations (e.g., 500ng/10^5 cells/mL) . After treatment, media should be collected and stored at -80°C prior to analysis. Commercial ELISA kits specifically designed for CCL11 detection provide standardized quantification methods .

When examining signaling pathways activated by CCL11, both in silico and in vitro approaches yield complementary insights. Computational tools like PseudoCell can predict potential molecular interactions and dynamic network responses to CCL11 stimulation at varying concentrations (using Node Activation Frequencies of 0% [control], 12.5% [low], 25% [medium], and 50% [high]) . These predictions should then be validated experimentally through techniques like flow cytometry, Western blotting, or immunofluorescence to confirm pathway activation.

How can CCL11-induced chemotaxis be quantitatively assessed in research settings?

Quantitative assessment of CCL11-induced chemotaxis requires careful experimental design and appropriate detection methods. A standard approach involves using transwell migration assays with CCR3-expressing cells, such as transfected BaF3 mouse pro-B cells . In this system, varying concentrations of recombinant mouse CCL11 (typically ranging from 1-100 ng/mL) are placed in the lower chamber, while cells are added to the upper chamber. Following incubation (usually 2-4 hours at 37°C), migrated cells can be quantified using Resazurin fluorescence detection .

For data analysis, researchers should:

• Construct dose-response curves by plotting chemotaxis against CCL11 concentration

• Calculate EC50 values (effective concentration inducing 50% of maximal migration)

• Compare experimental treatments to positive controls (known chemotactic factors) and negative controls (buffer alone)

• Consider chemotactic index calculations (ratio of cells migrating toward CCL11 versus random migration)

This methodological approach enables precise quantification of CCL11's chemotactic potency and facilitates comparison between experimental conditions or different CCL11 preparations .

What protocols are recommended for studying CCL11's effects on DNA damage and cellular senescence?

To investigate CCL11's impact on DNA damage and cellular senescence, researchers should implement multi-parameter analysis approaches. For DNA damage assessment, flow cytometry using phospho-specific antibodies against γH2AX and p-p53 provides quantitative measurements of DNA damage signaling activation .

A recommended protocol includes:

• Seed human lung fibroblasts (e.g., MRC-5) at 10×10^4 cells/mL

• Treat with high-dose rhCCL11 (500ng/10^5 cells/mL) for defined timepoints (2h and 24h)

• Fix and permeabilize cells for intracellular staining

• Stain with fluorophore-conjugated antibodies against γH2AX and p-p53

• Analyze by flow cytometry using appropriate laser configurations

• Quantify both percentage of positive cells and mean fluorescence intensity

For senescence studies, complementary assays should be performed, including:

• Senescence-Associated β-Galactosidase (SA-β-Gal) staining

• Assessment of senescence-associated secretory phenotype (SASP) markers like IL-6

• Analysis of cell cycle arrest through flow cytometry

• Evaluation of morphological changes associated with senescence

These combined approaches provide comprehensive characterization of CCL11's influence on cellular senescence pathways .

How should researchers design experiments to study CCL11 expression in tissue samples?

For effective analysis of CCL11 expression in tissue samples, immunohistochemistry (IHC) provides valuable spatial information. Based on established protocols, researchers should use perfusion-fixed frozen tissue sections for optimal results . The recommended staining procedure involves:

• Incubate sections with primary antibody (e.g., Goat Anti-Mouse CCL11/Eotaxin Antibody) at 15 μg/mL concentration overnight at 4°C

• Apply appropriate detection system (e.g., Anti-Goat HRP-DAB Cell & Tissue Staining Kit)

• Counterstain with hematoxylin for structural context

• Perform appropriate controls (isotype control, secondary antibody only)

• Analyze for cytoplasmic staining patterns, as CCL11 typically localizes to the cytoplasm

For quantitative tissue analysis, researchers may complement IHC with:

• Laser capture microdissection followed by qPCR or proteomics

• Multiplexed immunofluorescence to co-localize CCL11 with cell-type markers

• Digital image analysis for quantification of staining intensity and distribution

These approaches enable comprehensive characterization of CCL11 expression patterns within the complex tissue microenvironment .