Recombinant Mouse C-C motif chemokine 5 protein (Ccl5) (Active)

What is CCL5/RANTES and what are its primary functions in mouse models?

CCL5, also known as RANTES (Regulated upon Activation, Normal T cell Expressed and presumably Secreted), is an 8 kDa beta-chemokine that plays a fundamental role in inflammatory immune responses primarily through its ability to attract and activate leukocytes. In mouse models, CCL5 functions as a chemoattractant for eosinophils, monocytes, and lymphocytes, with demonstrated activity through CCR1, CCR3, CCR4, and CCR5 receptors .

Mouse CCL5 is particularly valuable in research as it shares 100% amino acid sequence identity with rat CCL5 and between 75-88% with canine, cotton rat, feline, and human CCL5, making it suitable for cross-species experimental designs . The protein is secreted by numerous cell types at inflammatory sites, making it a key mediator in various inflammatory conditions.

How should recombinant mouse CCL5 be reconstituted and stored for optimal activity?

For optimal experimental outcomes, reconstitution protocols differ based on carrier status:

For carrier-containing preparations (with BSA):

• Reconstitute lyophilized protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• Use immediately or store in working aliquots at -20°C to -80°C

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

For carrier-free preparations:

• Reconstitute at 100 μg/mL in sterile PBS

• Similarly, aliquot and store at -20°C to -80°C

The stability of the reconstituted protein varies by application, with optimal activity typically maintained for 1-3 months when properly stored. For critical experiments, freshly reconstituted protein is recommended to ensure maximum bioactivity.

What are the validated methods for assessing CCL5 biological activity in vitro?

Two established methodologies have been validated for determining the biological activity of recombinant mouse CCL5:

Chemotaxis assay with BaF/3 cells:

• Measures CCL5's ability to induce chemotaxis in BaF/3 cells (mouse pro-B cell line) transfected with appropriate chemokine receptors

• The effective dose (ED50) for this assay typically ranges from 1.5-9 ng/mL

• Quantification involves counting migrated cells in a modified Boyden chamber setup

Human monocyte chemotaxis assay:

• Measures CCL5's ability to chemoattract 2-day cultured human monocytes

• The ED50 for this effect ranges from 30-100 ng/mL

• Demonstrates cross-species activity relevant for translational research

When designing CCL5 functional experiments, appropriate positive and negative controls should be included to account for baseline migration.

How can CCL5 be effectively used in neuroinflammation and brain injury research models?

Recent studies have established critical protocols for applying CCL5 in neurological research contexts:

For traumatic brain injury (TBI) models:

• CCL5 has demonstrated essential roles in axonogenesis and neuronal restoration following brain injury

• Administration methods include:

  • Direct intracerebroventricular injection (typically 100-500 ng in 2-5 μL)

  • Local application to injury site using controlled-release hydrogels

  • Systemic administration (10-50 μg/kg, i.p.) for blood-brain barrier penetration studies

For memory and cognitive function studies:

• CCL5 promotion of bioenergy metabolism has been shown crucial for hippocampal synapse complex and memory formation

• Experimental approaches include:

  • In vivo administration followed by behavioral testing (Morris water maze, novel object recognition)

  • Ex vivo hippocampal slice preparations treated with recombinant CCL5 (10-100 ng/mL)

  • Primary neuronal cultures for signaling pathway analysis

Research has demonstrated that CCL5 works through GPX1 activation to protect hippocampal memory function after mild traumatic brain injury, suggesting potential therapeutic applications .

What are the key methodological considerations when studying CCL5's role in bone biology and osteoclastogenesis?

Studying CCL5 in bone biology requires specialized techniques addressing both molecular and cellular aspects:

For osteoclast differentiation and function studies:

• In vitro osteoclastogenesis assays using bone marrow-derived macrophages treated with recombinant CCL5 (10-100 ng/mL) alongside RANKL and M-CSF

• Functional analysis through pit formation assays on dentine slices or calcium phosphate substrates

• Real-time PCR analysis of osteoclast markers (TRAP, Cathepsin K, OSCAR)

For in vivo bone homeostasis models:

• CCL5-knockout mice exhibit impaired bone formation and increased osteoclastogenesis

• Methodological approaches include:

  • μCT analysis of trabecular and cortical bone parameters

  • Histomorphometric analysis of bone formation and resorption rates

  • In vivo calcein double labeling to measure dynamic bone formation

The HIV co-receptor CCR5 has been shown to regulate osteoclast function, highlighting the importance of investigating receptor-specific mechanisms when studying CCL5 in bone biology .

What techniques are optimal for investigating CCL5 oligomerization and its functional consequences?

CCL5 oligomerization represents a crucial regulatory mechanism affecting its biological functions:

Analytical techniques to study oligomerization:

• Size exclusion chromatography to separate monomeric, dimeric, and higher-order oligomeric forms

• Chemical cross-linking followed by SDS-PAGE analysis

• Analytical ultracentrifugation for molecular weight determination

• Dynamic light scattering for hydrodynamic radius measurement

Functional assessment of oligomerization states:

• Site-directed mutagenesis of residues involved in oligomerization

• Comparison of wild-type CCL5 vs. oligomerization-deficient mutants in:

  • GAG binding assays (heparin affinity chromatography)

  • Solid-phase binding assays with immobilized glycosaminoglycans

  • Leukocyte adhesion under flow conditions

How can researchers address inconsistent results when using recombinant CCL5 in chemotaxis assays?

Troubleshooting variable chemotaxis results requires systematic evaluation of multiple factors:

Common sources of variability and solutions:

For quantitative comparison between experiments, normalization to a standard positive control is essential. Additionally, researchers should consider that different cell types exhibit varying ED50 values, as evidenced by the difference between BaF/3 cells (ED50: 1.5-9 ng/mL) and human monocytes (ED50: 30-100 ng/mL) .

What considerations are important when interpreting CCL5 knockout or neutralization studies in complex disease models?

Interpreting data from CCL5 perturbation studies requires awareness of several methodological challenges:

Key considerations:

• Compensation mechanisms: Other chemokines, particularly within the CC chemokine family, may compensate for CCL5 deficiency

• Receptor promiscuity: CCL5 signals through multiple receptors (CCR1, CCR3, CCR4, CCR5), each contributing differently to phenotypes

• Tissue-specific effects: CCL5 function varies across tissues, as demonstrated in studies of bone formation, brain injury, and inflammation

• Temporal dynamics: Acute vs. chronic CCL5 depletion may yield different results due to adaptation mechanisms

Methodological approaches to improve interpretation:

• Combined knockout/neutralization of CCL5 with related chemokines

• Receptor-specific antagonism to dissect pathway contributions

• Tissue-specific conditional knockout models

• Inducible systems for temporal control of CCL5 expression

Research has shown that CCL5-knockout mice exhibit impaired bone formation and increased osteoclastogenesis, demonstrating the importance of this chemokine in bone homeostasis beyond its classical inflammatory roles .

How should researchers approach comparative studies between mouse and human CCL5 for translational research?

Translational research involving CCL5 requires careful consideration of cross-species similarities and differences:

Sequence and structural considerations:

• Mouse CCL5 shares 75-88% amino acid sequence identity with human CCL5

• Conserved functional domains enable cross-species activity in many assays

• Subtle structural differences may affect receptor binding affinities and signaling outcomes

Experimental approaches for translational studies:

• Parallel testing of mouse and human CCL5 on cells from both species

• Dose-response comparisons to identify potential sensitivity differences

• Species-specific receptor antagonism to identify divergent signaling pathways

• Humanized mouse models for studying human-specific effects

Key experimental considerations:

• Document species-specific ED50 values for each assay system

• Account for differences in receptor expression patterns between species

• Consider potential differences in post-translational modifications

• Validate findings in primary cells from both species when possible

Human and mouse CCL5 exhibit cross-species activity on cells from both species, making comparative studies feasible, but researchers should remain aware that quantitative differences in potency and receptor preference may exist .

How can researchers effectively utilize CCL5 in cancer immunology and tumor microenvironment studies?

CCL5's emerging role in cancer biology offers several experimental approaches:

For tumor microenvironment modeling:

• Co-culture systems with cancer cells and CCL5-producing stromal cells

• 3D organoid cultures with controlled CCL5 gradients

• In vivo tumor models comparing wild-type vs. CCL5-knockout backgrounds

Mechanistic studies in cancer progression:

• CCL5 has been demonstrated as essential for high-grade glioma growth regulatory circuits critical for mesenchymal glioblastoma survival

• Breast cancer progression is influenced by CCL5 through:

  • Attraction of pro-inflammatory macrophages

  • Direct effects on tumor cells

  • Modification of stromal and vascular components

Experimental readouts:

• Flow cytometry for immune cell infiltration profiling

• Histological assessment of tumor angiogenesis and invasion

• Real-time cell migration tracking in response to CCL5 gradients

• Gene expression analysis of CCL5-induced pathways in tumor cells

These approaches can help elucidate the dual roles of CCL5 in both promoting and inhibiting tumor growth, depending on the cancer type and microenvironment context.

What are the current methodologies for studying CCL5's role in vascular biology and angiogenesis?

CCL5 has emerging functions in vascular biology that can be studied through various approaches:

In vitro angiogenesis models:

• Endothelial tube formation assays with recombinant CCL5 (10-100 ng/mL)

• Endothelial cell migration and proliferation assays

• Aortic ring explant cultures for ex vivo sprouting analysis

In vivo vascular models:

• Matrigel plug assays with incorporated CCL5

• Hindlimb ischemia models with CCL5 delivery systems

• Retinal vascularization in developmental models

Advanced delivery systems:

• RANTES-loaded polysaccharide-based microparticles have shown pro-angiogenic effects in mouse ischemia therapy

• Controlled release formulations can provide sustained CCL5 delivery to ischemic tissues

Analytical methods:

• Laser Doppler perfusion imaging for blood flow assessment

• Micro-CT angiography for 3D vascular network analysis

• Immunohistochemical analysis of angiogenic markers (CD31, SMA, Desmin)

Research has demonstrated CCL5's importance in tissue repair through mesenchymal stem cell support of ischemic regions, suggesting therapeutic potential for tissue engineering applications .

What innovative approaches are being developed to study CCL5 post-translational modifications and their functional consequences?

Post-translational modifications (PTMs) significantly impact CCL5 function but require specialized techniques:

Analytical methods for PTM identification:

• High-resolution mass spectrometry to identify and quantify modifications

• Site-specific antibodies against modified CCL5 forms

• 2D gel electrophoresis for charge variant separation

• Lectin affinity chromatography for glycosylated variants

Functional assessment of modified CCL5:

• Comparison of native vs. CD26/DPPIV-processed CCL5 (N-terminal truncation)

  • The removal of two N-terminal residues generates a chemotaxis inhibitor that more effectively blocks certain HIV-1 infections

• Analysis of nitrated or citrullinated CCL5 in inflammatory conditions

• Evaluation of CCL5 glycosylation patterns in different disease states

Generation of modified CCL5 for research:

• Enzymatic modification with purified modifying enzymes (DPP4, PAD, etc.)

• Chemical synthesis of specifically modified CCL5 variants

• Expression systems with co-transfected modifying enzymes

These approaches enable researchers to understand how enzymatic processing and other modifications alter CCL5's receptor specificity, oligomerization properties, and biological functions in different physiological and pathological contexts.