Recombinant Mouse Fractalkine protein (Cx3cl1), partial (Active)

What is the molecular structure of Recombinant Mouse Fractalkine (Cx3cl1) protein, and how does it compare to the native form?

Recombinant Mouse Fractalkine protein typically comprises only the chemokine domain (approximately amino acids 22-105 or 25-105) of the native protein. While native Fractalkine exists in two forms—a membrane-bound protein (~95 kDa) tethered by a mucine-like stalk and a soluble factor (~70 kDa) released upon cleavage—the recombinant protein represents just the chemokine domain with a molecular mass of approximately 8.7-9.5 kDa . This non-glycosylated polypeptide chain contains 76-84 amino acids and lacks the mucin-like stalk present in the membrane-bound form .

The amino acid sequence of commercially available Recombinant Mouse Cx3cl1 is typically:
QHLGMTKCEIMCGKMTSRIPVALLIRYQLNQESCGKRAIVLETTQHRRFCADPKEKWVQDAMKHLDHQAAALTKNG

How does the recombinant partial Mouse Cx3cl1 maintain biological activity despite lacking the mucin-like stalk?

The chemokine domain alone is sufficient for receptor binding and activation. Functional assays demonstrate that the E. coli-derived recombinant chemokine domain maintains high biological activity, with ED50 values typically less than 0.5 ng/mL in cell proliferation assays using human peripheral blood lymphocytes (PBL), corresponding to a specific activity greater than 2000 IU/mg . This indicates that while the mucin-like stalk is important for membrane tethering in native contexts, the chemokine domain independently retains receptor-binding properties essential for biological function .

What are the optimal methodologies for using Recombinant Mouse Cx3cl1 in chemotaxis assays?

For chemotaxis assays, the recommended methodology includes:

• Cell preparation: Use CX3CR1-expressing cells such as microglia, monocytes, or CX3CR1-transfected cell lines (e.g., BaF3 mouse pro-B cells transfected with human CX3CR1) .

• Assay setup: A standard transwell migration system (e.g., millicell PCF 8 μm insert) with Recombinant Mouse Cx3cl1 added to the lower chamber at concentrations ranging from 1-100 ng/mL. The optimal concentration for chemotaxis is typically around 30 ng/mL .

• Quantification: After 3-4 hours incubation, migrated cells can be quantified by fluorescence (if cells express fluorescent proteins), by DAPI nuclear staining, or by metabolic assays such as Resazurin .

• Controls: Include both negative controls (media without Cx3cl1) and positive controls (known chemoattractants like SDF-1 or MCP-1) to validate the assay system .

In validation studies, dose-dependent migration responses typically show a bell-shaped curve, with maximal migration at 10-50 ng/mL of Recombinant Mouse Cx3cl1 .

How can researchers effectively validate the cross-species reactivity between Mouse Cx3cl1 and human CX3CR1?

Cross-species reactivity between Mouse Cx3cl1 and human CX3CR1 can be validated using the following approaches:

• Transmigration assays: Culture bone marrow-derived macrophages from CX3CR1-knockout mice and transfect them with expression vectors containing human CX3CR1 variants (reference V249/T280 or variant I249/M280). Expose these cells to different concentrations of soluble recombinant Mouse Fractalkine (1-100 ng/mL) in a transwell system and quantify migrated cells .

• Calcium mobilization assays: Measure intracellular calcium flux in human CX3CR1-expressing cells after stimulation with Mouse Cx3cl1 .

• Binding assays: Use radiolabeled or fluorescently-labeled Mouse Cx3cl1 to measure direct binding to human CX3CR1-expressing cells .

Research demonstrates that human CX3CR1 receptors can respond to Mouse Fractalkine, but with varying efficiency depending on the human receptor variant. The reference human receptor (V249/T280) signals effectively in response to Mouse Fractalkine, while the variant human receptor (I249/M280) shows blunted responses .

How can Recombinant Mouse Cx3cl1 be utilized to investigate microglial physiology in neuroinflammation models?

Recombinant Mouse Cx3cl1 serves as a valuable tool for dissecting microglial responses in neuroinflammation through several methodological approaches:

• Microglia isolation and stimulation: Primary microglia can be isolated from mouse brain tissue and stimulated with Recombinant Mouse Cx3cl1 to study:

  • Calcium mobilization (within minutes of application)

  • Cytoskeletal rearrangement

  • Migratory behavior

  • Cytokine production profiles

  • Gene expression changes

• Ex vivo slice cultures: Brain slices can be treated with Recombinant Mouse Cx3cl1 to observe microglial dynamics in a preserved tissue architecture using time-lapse microscopy .

• Comparative studies with CX3CR1-deficient models: Experiments comparing wild-type microglia with CX3CR1-knockout microglia can evaluate receptor-dependent responses to Recombinant Mouse Cx3cl1 .

• EAE (Experimental Autoimmune Encephalomyelitis) models: Researchers can manipulate the Fractalkine/CX3CR1 axis using Recombinant Mouse Cx3cl1 in combination with MOG35-55 immunization to study microglial contributions to neuroinflammation .

Research findings indicate that Fractalkine signaling critically regulates microglial properties during normal physiological conditions. In neuroinflammatory conditions, basal fractalkine levels in wild-type mice show approximately fivefold increase during EAE-induced disease, while CX3CR1-deficient mice show elevated baseline levels that remain unchanged during disease progression .

What methodological approaches can be employed to study the role of Cx3cl1-CX3CR1 signaling in osteoclastogenesis?

To investigate Cx3cl1-CX3CR1 signaling in osteoclastogenesis, researchers can employ the following methodologies:

• Osteoclast precursor (OCP) culture systems:

  • Isolate CD11bhighCD115+ OCPs from bone marrow

  • Culture OCPs on immobilized Recombinant Mouse Cx3cl1 prior to RANKL stimulation

  • Assess osteoclast formation through TRAP staining and counting multinucleated cells

• Gene expression analysis:

  • Monitor expression of osteoclast differentiation markers (Nfatc1, Calcr, Ctsk)

  • Track dynamic changes in Cx3cr1 and Rank (Tnfrsf11a) expression during differentiation using qRT-PCR

  • Studies show that growth on immobilized Cx3cl1 increases expression of both Cx3cr1 and Rank transcripts, but following RANKL stimulation, OCPs rapidly downregulate Cx3cr1 expression

• Survival and differentiation assays:

  • Measure OCP survival using Annexin V/PI staining

  • Quantify CD11bhighCD115+ OCP numbers using flow cytometry

  • Research indicates that growth on immobilized Cx3cl1 prior to RANKL stimulation increases CD11bhighCD115+ OCP number and enhances their survival and differentiation potential

• In vivo models of bone loss:

  • RANKL-induced bone loss model with anti-Cx3cl1 monoclonal antibody pretreatment

  • µCT analysis of bone parameters

  • Studies demonstrate that anti-Cx3cl1 mAb pretreatment significantly inhibits RANKL-dependent bone loss

What are critical quality control parameters for ensuring reproducible results with Recombinant Mouse Cx3cl1?

To ensure reproducible results when working with Recombinant Mouse Cx3cl1, researchers should verify the following critical parameters:

• Purity assessment: Confirm protein purity is >97% by SDS-PAGE and/or HPLC analysis to avoid contamination with bacterial proteins that may cause confounding effects .

• Endotoxin testing: Verify that endotoxin levels are below 0.1 EU/µg of protein using LAL method to prevent non-specific immune activation in cellular assays .

• Biological activity validation: Confirm specific activity using standardized assays such as:

  • Cell proliferation assays with human peripheral blood lymphocytes (expected ED50 <0.5 µg/ml)

  • Chemotaxis assays using CX3CR1-expressing cells (expected dose-dependent response)

• Reconstitution protocol adherence: Reconstitute lyophilized protein according to manufacturer specifications to maintain activity:

  • Use sterile buffers (typically PBS, pH 7.4)

  • Avoid excessive vortexing

  • Allow complete reconstitution before use

• Storage conditions: Maintain proper storage to prevent activity loss:

  • Store lyophilized protein at -20°C to -70°C

  • Store reconstituted protein at 2-8°C for short-term (1 month) or -20°C to -70°C (3 months) for long-term use

  • Avoid repeated freeze-thaw cycles

How can researchers address experimental variability when comparing studies using different Recombinant Mouse Cx3cl1 preparations?

When comparing studies using different Recombinant Mouse Cx3cl1 preparations, researchers should implement the following strategies to address experimental variability:

• Standardization of activity units: Convert protein concentrations to activity units (IU/mg) based on standardized bioassays to normalize for preparation differences between manufacturers .

• Internal calibration curves: Generate dose-response curves with each new lot of Recombinant Mouse Cx3cl1 to identify the optimal working concentration for specific experimental systems .

• Reference standards: Include a well-characterized laboratory reference standard across experiments to normalize results from different commercial preparations .

• Comprehensive reporting: Document and report the following parameters in publications:

  • Manufacturer and catalog number

  • Lot number and production date

  • Expression system (typically E. coli)

  • Amino acid range included in the recombinant protein

  • Reconstitution buffer composition

  • Storage conditions and duration before use

• Validation with neutralizing antibodies: Confirm specificity of observed effects by using anti-Cx3cl1 neutralizing antibodies such as Goat Anti-Mouse CX3CL1/Fractalkine Chemokine Domain Antigen Affinity-purified Polyclonal Antibody to block activity in parallel experiments .

Research has shown that the ND50 (neutralization dose) of anti-Cx3cl1 antibodies is typically 0.3-1.5 µg/mL in the presence of 30 ng/mL Recombinant Mouse Cx3cl1 .

How can Recombinant Mouse Cx3cl1 and anti-Cx3cl1 antibodies be used to investigate therapeutic potential in fibrotic disorders?

Recent research reveals promising applications of Recombinant Mouse Cx3cl1 and anti-Cx3cl1 antibodies in investigating potential therapies for fibrotic disorders:

• Fibroblast response studies:

  • Treat human dermal fibroblasts with Recombinant Mouse Cx3cl1 with or without anti-Cx3cl1 mAb

  • Measure expression of fibrosis markers (type I collagen, fibronectin 1)

  • Studies show that anti-Cx3cl1 mAb treatment significantly inhibits TGF-β1-induced expression of type I collagen and fibronectin 1 in human dermal fibroblasts

• Multi-model validation approach:

  • Test anti-Cx3cl1 mAb therapy in multiple mouse models of fibrosis:

    • Bleomycin-induced fibrosis

    • Growth factor-induced fibrosis

    • Scleroderma-chronic graft-versus-host disease (Scl-cGVHD)

  • Research demonstrates anti-mouse Cx3cl1 mAb efficiently suppresses skin inflammation and fibrosis across these models

• Organ-specific effects assessment:

  • Investigate effects on both skin and lung fibrosis

  • Measure inflammatory cell infiltration, collagen deposition, and fibrosis-related gene expression

  • Recent findings indicate anti-Cx3cl1 mAb therapy could be a rational therapeutic approach for fibrotic disorders such as human systemic sclerosis (SSc) and Scl-cGVHD

• Mechanisms of action studies:

  • Investigate Cx3cl1-induced signaling in target cells using Recombinant Mouse Cx3cl1

  • Determine how anti-Cx3cl1 antibodies interrupt pathological signaling cascades

  • Measure fractalkine levels in tissue extracts to correlate with disease severity

What are the methodological considerations when using transgenic mouse models expressing human CX3CR1 variants in combination with Recombinant Mouse Cx3cl1?

When working with transgenic mouse models expressing human CX3CR1 variants and Recombinant Mouse Cx3cl1, researchers should consider these methodological approaches:

• Cross-species signaling validation:

  • Confirm responsiveness of human CX3CR1 receptors to mouse fractalkine in vitro using bone marrow-derived macrophages electroporated with human CX3CR1 expression vectors

  • Perform dose-response transmigration assays (1-100 ng/mL mouse Cx3cl1)

  • Research shows the reference human receptor (V249/T280) signals effectively in response to mouse Cx3cl1, while the variant receptor (I249/M280) displays blunted responses

• Receptor expression confirmation:

  • Validate human CX3CR1 expression at transcript levels in sorted microglial cells from transgenic mice

  • Compare expression levels to wild-type and CX3CR1-knockout controls

  • Studies confirm that mice expressing the human CX3CR1 I249/M280 variant exhibit CX3CR1 mRNA levels comparable to wild-type mice

• Functional response assessment:

  • Compare cellular responses to Recombinant Mouse Cx3cl1 between wild-type mice, CX3CR1-knockout mice, and human CX3CR1 variant mice

  • Measure parameters such as cell migration, calcium mobilization, and cytokine production

  • Research indicates that human CX3CR1 I249/M280 variant mice show intermediate fractalkine levels in naïve conditions that appear sustained upon EAE induction

• Disease model phenotyping:

  • Characterize how human CX3CR1 variants respond to mouse Cx3cl1 in various disease models

  • Measure fractalkine levels, inflammatory markers, and tissue-specific pathology

  • Studies show that mice expressing the human CX3CR1 I249/M280 variant exhibit similar defects in CNTF production and neuronal cell loss as CX3CR1-knockout mice during chronic EAE

These methodological considerations enable researchers to use transgenic models to uniquely define the role of human CX3CR1 variants in various disease contexts while leveraging the research utility of Recombinant Mouse Cx3cl1.