Recombinant Rat Eotaxin protein (Ccl11) (Active)

What is the basic structure of Recombinant Rat Eotaxin (CCL11)?

Rat Eotaxin belongs to the platelet factor-4 family of chemokines and is also referred to as Eotaxin-1 or CCL11. The recombinant protein derived from E. coli has a molecular weight of approximately 8 kDa. The protein is typically supplied as a lyophilized powder with >95% purity as determined by SDS-PAGE. Rat Eotaxin shares approximately 60% sequence identity with human CCL11 .

What are the primary biological functions of Rat Eotaxin (CCL11)?

Rat Eotaxin functions as a potent chemoattractant primarily for eosinophils. It induces substantial accumulation of eosinophils in tissues without significantly affecting neutrophil accumulation. The primary activity can be measured through chemotaxis assays using human peripheral blood eosinophils at a concentration range of 0.1-20 ng/mL. The protein binds to CCR3, a G-protein-coupled receptor selectively expressed on eosinophils and several other cell types .

How does Rat CCL11 compare structurally and functionally to human and guinea pig CCL11?

Rat CCL11 shares approximately 60% amino acid sequence identity with human and guinea pig CCL11. Despite these differences, all three species variants function as potent eosinophil chemoattractants. Human CCL11 specifically consists of a 97 amino acid precursor from which 23 amino acid residues are cleaved to generate the 74 amino acid mature protein. The rat variant maintains similar functional properties while exhibiting species-specific structural variations that may impact cross-species reactivity in experimental settings .

What is the recommended protocol for reconstituting lyophilized Rat CCL11?

For optimal reconstitution, perform a quick spin of the vial containing lyophilized Rat CCL11, then add distilled water to achieve a concentration not less than 0.1 mg/mL. This initial solution can then be further diluted into other appropriate buffers depending on the experimental requirements. When properly reconstituted, the protein demonstrates chemotactic activity at concentrations of 0.1-20 ng/mL in eosinophil migration assays .

What are the optimal storage conditions for maintaining Rat CCL11 activity?

The lyophilized protein remains stable for at least one year when stored at -70°C. After reconstitution, working aliquots can be stored at 2-8°C for one month or at -20°C for six months with a carrier protein without detectable loss of activity. Importantly, repeated freeze/thaw cycles should be avoided as they can significantly compromise protein integrity and biological activity .

How can researchers verify the activity of Rat CCL11 preparations before experimental use?

Functional activity can be verified using a chemotaxis assay with human peripheral blood eosinophils. The expected effective concentration range is 0.1-20 ng/mL. Additionally, researchers can verify receptor binding using CCR3-transfected cell lines and calcium flux assays. For structural integrity assessment, SDS-PAGE can confirm the expected molecular weight of approximately 8 kDa and evaluate purity levels (expected >95%) .

Which cellular receptors mediate Rat CCL11 biological effects?

CCR3 serves as the primary receptor for Rat CCL11. This G-protein-coupled receptor is predominantly expressed on eosinophils but has also been identified on several other cell types including vascular smooth muscle cells (SMCs) and neutrophils. The CCL11-CCR3 interaction triggers intracellular signaling cascades that culminate in directional cell migration. Blocking CCR3 with neutralizing antibodies significantly inhibits CCL11-induced chemotaxis, confirming the receptor's central role in mediating CCL11 functions .

How does CCL11 signaling differ between immune and non-immune cell types?

While CCL11 primarily signals through CCR3 in both immune and non-immune cells, the downstream effects vary substantially. In eosinophils, CCL11 induces robust chemotaxis and calcium flux, promoting inflammatory responses. In vascular smooth muscle cells, CCL11 induces concentration-dependent migration with maximum effect at approximately 100 ng/mL, but notably does not stimulate proliferation. This suggests cell-type-specific signaling pathways downstream of CCR3 activation. In experimental settings, CCR3 antibodies effectively block CCL11-induced SMC migration, further confirming receptor specificity across cell types .

What is the relationship between CCL11-CCR3 signaling and calcium flux in target cells?

CCL11 binding to CCR3 induces calcium flux in eosinophils and CCR3-transfected cells, which represents an early signaling event critical for subsequent cellular responses. This calcium mobilization serves as a useful experimental readout for CCR3 activation. Similar chemokines that bind CCR3, such as Eotaxin-3/CCL26, can cross-desensitize cells to CCL11, suggesting overlapping signaling mechanisms. Experimental protocols monitoring calcium flux typically employ fluorescent calcium indicators and flow cytometry or plate-based fluorescence methods to quantify responses .

How can Rat CCL11 be utilized in pulmonary fibrosis research models?

Rat CCL11 plays a significant role in pulmonary fibrosis models, particularly those induced by bleomycin treatment. Research protocols typically involve administering bleomycin (approximately 0.02 U/mouse) followed by assessment of CCL11 expression and function. Studies with CCL11-deficient mice have demonstrated significantly reduced pulmonary fibrosis and diminished expression of profibrotic cytokines such as transforming growth factor-β1 (TGF-β1). Conversely, increased lung expression of CCL11 enhances bleomycin-induced lung fibrosis. For mechanistic studies, neutralizing CCR3 antibodies can be administered to evaluate the specific contribution of CCL11-CCR3 signaling to fibrotic processes .

What experimental approaches can evaluate CCL11's role in vascular pathologies?

CCL11 and its receptor CCR3 are abundantly expressed in atheromatous plaques and after arterial injury, suggesting important roles in vascular pathologies. In experimental settings, researchers can use modified Boyden chamber assays to assess CCL11-induced vascular smooth muscle cell (SMC) migration, with maximum effects typically observed at 100 ng/mL. Scrape-wound assays provide an alternative method for evaluating SMC migration. CCL11's effects on vascular function can be specifically validated using CCR3-neutralizing antibodies as experimental controls. Importantly, CCL11 does not affect SMC proliferation, allowing researchers to distinguish between migratory and proliferative responses in vascular disease models .

How can interaction studies between CCL11 and other chemokines be designed?

Experimental designs to investigate interactions between CCL11 and other chemokines should consider cross-desensitization studies, where cells are sequentially exposed to different chemokines to assess receptor desensitization. For example, CCL11 and CCL26 (Eotaxin-3) both signal through CCR3, and prior exposure to one can affect cellular responses to the other. Researchers can design dose-response and time-course experiments using chemotaxis assays, calcium flux measurements, or receptor internalization studies to quantify these interactions. Additionally, competitive binding assays using labeled chemokines can directly measure receptor occupation and displacement .

What in vivo gene expression approaches can manipulate CCL11 levels for functional studies?

Adenoviral vector-based gene delivery systems have been successfully used to modulate CCL11 expression in vivo. In experimental protocols, adenoviral vectors containing CCL11 cDNA (typically administered at 0.5-10 × 10^8 pfu) are delivered through oral instillation, resulting in significant overexpression. This approach yields approximately 3-4 fold increases in CCL11 protein levels (5.8-7.1 ng/lung compared to 1.6-1.7 ng/lung in controls). For timing considerations, maximal gene expression typically occurs between 7-10 days after administration. This approach is particularly effective for studying CCL11's role in pulmonary pathologies, as the technique preferentially targets epithelial cells .

How can researchers quantitatively assess CCL11 and CCR3 expression in experimental samples?

Quantitative assessment of CCL11 and CCR3 expression can be performed using multiple complementary approaches. For mRNA quantification, quantitative RT-PCR using TaqMan assays allows precise measurement of transcript levels, with data typically normalized to housekeeping genes like GAPDH and expressed as 2^(-ΔCT). Protein levels can be quantified using ELISA (typically with detection limits in the pg/mL range), with results expressed as ng/lung or pg/mL depending on sample type. For cellular localization studies, immunohistochemistry with specific antibodies against CCL11 and CCR3 can identify expression patterns in tissue sections, particularly in disease models like arterial injury where expression patterns change significantly over time .

What are the critical considerations when designing studies comparing different species variants of CCL11?

When designing comparative studies across species variants of CCL11 (human, rat, mouse, guinea pig), researchers must account for sequence variations (approximately 60% identity between species) that may affect cross-reactivity of detection reagents and functional outcomes. Species-specific receptors should be considered, as receptor-ligand affinities may vary. Experimental controls should include species-matched positive controls and dose-response curves for each variant. For functional comparisons, standardized assays such as chemotaxis of CCR3-transfected cell lines allow direct comparison of potency and efficacy. When interpreting results, researchers should consider evolutionary conservation of CCL11 functions versus species-specific adaptations in receptor distribution and signaling pathways .

How can researchers address inconsistent results in CCL11-induced chemotaxis assays?

Inconsistent results in CCL11-induced chemotaxis assays can stem from multiple factors. First, verify protein activity by ensuring proper reconstitution in appropriate buffers and avoiding repeated freeze-thaw cycles. For eosinophil chemotaxis, the optimal concentration range is typically 0.1-20 ng/mL, while for smooth muscle cells, higher concentrations (up to 100 ng/mL) may be required for maximum effect. Cell preparation techniques significantly impact responsiveness - freshly isolated cells generally demonstrate better responses than cultured cells. Additionally, ensure that experimental cells express sufficient CCR3 by flow cytometry verification prior to experiments. Control experiments with CCR3-neutralizing antibodies can confirm specificity, and positive controls with established chemoattractants help validate assay functionality .

What strategies can minimize variability in CCL11 protein quantification from biological samples?

To minimize variability in CCL11 protein quantification from biological samples such as bronchoalveolar lavage fluid or tissue homogenates, implement standardized collection protocols that include protease inhibitors to prevent protein degradation. For ELISA-based detection, generate standard curves in the same matrix as experimental samples to account for matrix effects. Technical replicates (minimum triplicate) are essential, and samples with values outside the linear range of the standard curve should be appropriately diluted and re-analyzed. When comparing samples across experimental groups, process all samples simultaneously with the same reagent lots. For time-course studies, consider the stability of CCL11 under storage conditions and standardize freeze-thaw cycles. Finally, normalize protein measurements to total protein content or tissue weight to account for sampling variations .

How can researchers optimize CCL11 reconstitution to maintain maximum biological activity?

For optimal reconstitution that maintains maximum biological activity, begin with a quick centrifugation of the lyophilized protein vial to collect all material at the bottom. Add sterile distilled water to achieve a concentration not less than 0.1 mg/mL, allowing the protein to dissolve completely before gentle mixing. Avoid vigorous vortexing which can cause protein denaturation. For long-term storage of reconstituted protein, consider adding carrier proteins like BSA (0.1-1%) to prevent adsorption to storage vessels and maintain stability. Prepare single-use aliquots to avoid repeated freeze-thaw cycles and store at -20°C for up to six months or at 2-8°C for short-term use (up to one month). Prior to functional assays, perform a pilot dose-response experiment to verify activity within the expected concentration range (0.1-20 ng/mL for eosinophil chemotaxis) .