CX3CR1 Antibody

What is CX3CR1 and why is it important in research?

CX3CR1, also known as fractalkine receptor or G-protein coupled receptor 13 (GPR13), is a transmembrane protein belonging to the G protein-coupled receptor 1 (GPCR1) family. It is the sole member of the CX3C chemokine receptor subfamily and is encoded by the CX3CR1 gene in humans. Its significance stems from its critical role in inflammatory processes, cell migration, and tissue-specific immune responses. CX3CR1 is constitutively expressed in various hematopoietic-derived cells including T lymphocytes, natural killer cells, dendritic cells, monocytes, macrophages, microglia, and neutrophils, making it a valuable marker in immunology research . Recent studies have implicated the CX3CL1-CX3CR1 axis in fibrotic disorders, such as systemic sclerosis, highlighting its potential as a therapeutic target .

Which cell types express CX3CR1 and how can antibodies help identify them?

CX3CR1 is expressed in multiple cells of hematopoietic lineage, including:

• T lymphocytes

• Natural killer (NK) cells

• Dendritic cells

• B lymphocytes

• Mast cells

• Monocytes

• Macrophages

• Neutrophils

• Microglia

• Osteoclasts

• Thrombocytes

Anti-CX3CR1 antibodies enable identification of these cell populations through techniques like immunofluorescence, flow cytometry, and immunohistochemistry. For example, flow cytometry using anti-CX3CR1 antibodies can distinguish CX3CR1-expressing myeloid cells, as demonstrated in studies with the M1 mouse myeloid leukemia cell line . When combined with other lineage markers, CX3CR1 antibodies can help identify specific subpopulations in complex tissues like the central nervous system or inflammatory microenvironments .

What applications are CX3CR1 antibodies validated for?

CX3CR1 antibodies have been validated for multiple research applications:

Validation studies have confirmed specificity in multiple species, with antibodies detecting bands at approximately 40-50 kDa in western blot analysis, consistent with the predicted molecular weight of CX3CR1 .

How should I optimize CX3CR1 antibody staining protocols for immunofluorescence?

Optimizing CX3CR1 antibody staining requires careful consideration of several factors:

• Fixation: For membrane proteins like CX3CR1, a mild fixation protocol is recommended. Typically, 2-4% paraformaldehyde for 10-15 minutes preserves epitope accessibility while maintaining tissue architecture.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) often enhances CX3CR1 detection, particularly in formalin-fixed tissues.

• Blocking: Thorough blocking with 5-10% normal serum (matching the species of secondary antibody) with 0.1-0.3% Triton X-100 for 1-2 hours minimizes non-specific binding.

• Antibody concentration: Start with the manufacturer's recommended dilution (typically 1:100 for immunofluorescence) and optimize as needed .

• Incubation conditions: Overnight incubation at 4°C typically yields optimal staining with minimal background.

• Secondary antibody selection: Choose secondary antibodies with minimal cross-reactivity to the species being examined, and include appropriate controls to verify specificity.

For optimal results in rodent tissues, validated anti-CX3CR1 antibodies have demonstrated reliable performance at 1:100 dilution with standard blocking protocols .

What are the critical controls for validating CX3CR1 antibody specificity?

Rigorous validation of CX3CR1 antibody specificity requires multiple controls:

• Positive controls: Include known CX3CR1-expressing cells or tissues, such as:

  • Microglia in brain tissue

  • NK-92 human natural killer lymphoma cell line

  • M1 mouse myeloid leukemia cell line

• Negative controls:

  • Primary antibody omission

  • Isotype controls (e.g., Catalog # AB-108-C has been successfully used as an isotype control for CX3CR1 antibodies)

  • CX3CR1 knockout tissues when available

  • Cell lines known not to express CX3CR1

• Peptide competition: Pre-absorption of the antibody with the immunizing peptide should abolish specific staining.

• Multiple detection methods: Confirm expression using two or more techniques (e.g., western blot and immunostaining).

• Cross-validation with different antibody clones: Comparing results from different antibodies targeting distinct epitopes enhances confidence in specificity.

Research data supports this approach, as demonstrated in validation studies showing consistent detection patterns across multiple techniques .

How can I troubleshoot weak or non-specific CX3CR1 staining?

When encountering issues with CX3CR1 antibody staining, consider these solutions:

For weak signals:

• Increase antibody concentration incrementally

• Extend primary antibody incubation time (overnight at 4°C)

• Optimize antigen retrieval methods (try different buffers or retrieval times)

• Use signal amplification systems (e.g., biotin-streptavidin, tyramide signal amplification)

• Ensure samples are properly fixed and processed

• Check for protein degradation in older samples

For high background or non-specific staining:

• Increase blocking time and concentration

• Dilute antibody further

• Reduce detergent concentration in wash buffers

• Add 0.1-0.3% Triton X-100 to antibody diluent

• Include 1-5% BSA or normal serum in antibody diluent

• Increase washing steps and duration

• Ensure secondary antibody specificity

For inconsistent results:

• Standardize tissue processing protocols

• Consider that CX3CR1 expression can be modulated by inflammation, affecting detection levels

• Verify antibody storage conditions and avoid freeze-thaw cycles

• Check microscope settings for consistency across experiments

Published studies have successfully detected CX3CR1 in multiple tissue contexts using these optimization approaches .

How do CX3CR1 antibodies perform across different species and what are the cross-reactivity considerations?

CX3CR1 antibodies exhibit variable cross-reactivity depending on the epitope targeted and species homology:

When selecting antibodies for cross-species applications, consider:

• Epitope conservation: For the rat anti-CX3CR1 polyclonal antibody, the synthetic peptide immunogen is identical between mouse and rat sequences, showing 66.7% homology to human sequences . This explains its validated reactivity in both rodent species.

• Domain targeting: Antibodies targeting conserved domains (such as AF5825, which targets multiple conserved epitopes) often show better cross-reactivity between human and mouse samples .

• Validation method relevance: Antibodies validated by western blot may not necessarily work for immunohistochemistry in the same species due to differences in epitope accessibility.

To ensure reliability across species, preliminary validation experiments are strongly recommended before proceeding with full-scale studies.

How can CX3CR1 antibodies be used to study neuroinflammation and microglial function?

CX3CR1 antibodies are valuable tools for investigating neuroinflammation due to the high expression of CX3CR1 on microglia. Research applications include:

• Microglial identification: Anti-CX3CR1 antibodies can specifically label microglia in brain tissue, distinguishing them from other CNS cells.

• Activation state analysis: Changes in CX3CR1 expression correlate with microglial activation states. Quantitative analysis of staining intensity can reveal activation patterns in disease models.

• Co-localization studies: Combining CX3CR1 antibodies with markers for proinflammatory cytokines or activation markers provides insights into microglial phenotypes.

• In vivo tracking: Using CX3CR1 antibodies to identify microglia in tissue sections from experimental models allows tracking of migration and morphological changes.

• Therapeutic target validation: As reported in mouse models, the CX3CL1-CX3CR1 axis represents a potential therapeutic target for neuroinflammatory conditions .

Methodologically, when studying microglia, consider:

• Using thin sections (10-30 μm) to capture microglial processes

• Employing z-stack confocal microscopy for 3D reconstruction

• Combining with other markers (Iba1, TMEM119) for comprehensive characterization

• Quantifying morphological parameters (process length, branching) in addition to expression levels

These approaches have been validated in studies examining microglial responses in neurodegenerative disease models .

What role does the CX3CL1-CX3CR1 axis play in fibrotic disorders and how can antibodies help investigate this?

The CX3CL1-CX3CR1 axis has emerged as a critical pathway in fibrotic disorders, particularly in systemic sclerosis (SSc) and related conditions. Research findings reveal:

• Elevated serum biomarkers: Increased soluble CX3CL1 levels are observed in patients with severe SSc, interstitial lung disease, and digital ulcers .

• Therapeutic potential: Anti-CX3CL1 monoclonal antibody treatment significantly inhibits TGF-β1-induced expression of type I collagen and fibronectin in human dermal fibroblasts, suggesting a direct anti-fibrotic effect .

• Multi-organ benefits: Anti-CX3CL1 mAb therapy reduced both skin and lung fibrosis in sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) mouse models without apparent adverse events .

• Cellular mechanism: The therapeutic effects correlate with reduced tissue-infiltrating inflammatory cells, particularly CX3CR1-expressing macrophages and T cells, and decreased α-SMA-positive myofibroblasts in affected tissues .

CX3CR1 antibodies can be employed to investigate these mechanisms through:

• Immunohistochemical analysis to quantify CX3CR1-expressing cells in fibrotic tissues

• Flow cytometry to characterize infiltrating immune cell populations

• Co-localization studies with fibrosis markers like α-SMA and collagen

• In vitro studies examining the effects of CX3CL1/CX3CR1 blockade on fibroblast activation

These approaches have yielded valuable insights in preclinical studies, demonstrating that disruption of CX3CL1-CX3CR1 signaling may represent a rational therapeutic strategy for fibrotic disorders .

How do I design multi-color flow cytometry panels that include CX3CR1 for immune cell phenotyping?

Designing effective multi-color flow cytometry panels incorporating CX3CR1 requires strategic planning:

• Panel design considerations:

  • CX3CR1 is expressed at variable levels across immune cells, so pairing with lineage-defining markers is essential

  • Common combinations include:

    • CX3CR1/CD14/HLA-DR for monocyte subsets

    • CX3CR1/CD3/CD56 for NK and T cell populations

    • CX3CR1/CD11b/Ly6C for mouse myeloid populations

• Fluorophore selection:

  • Choose brighter fluorophores (PE, APC) for CX3CR1 when expression levels might be variable

  • Consider tandem dyes with minimal compensation requirements

  • Validated conjugates include PE-conjugated anti-goat IgG secondary antibodies used successfully with primary CX3CR1 antibodies

• Staining protocol optimization:

  • Surface staining: Perform on ice to minimize receptor internalization

  • Buffer selection: Include sodium azide to prevent endocytosis during staining

  • Titrate antibody concentration to achieve optimal signal-to-noise ratio

  • When using indirect staining approaches, include appropriate blocking steps

• Controls and analysis strategies:

  • Include isotype controls (such as Catalog # AB-108-C) to establish positive population boundaries

  • Use fluorescence-minus-one (FMO) controls for setting gates

  • Consider density plots rather than histograms for visualizing CX3CR1+ populations

• Validation approaches:

  • Confirm staining patterns with alternative techniques

  • Compare with known population frequencies from literature

  • Use cell sorting followed by functional assays to confirm identity

These approaches have been successfully employed in studies characterizing CX3CR1+ cell populations in various disease models and tissues .

How can CX3CR1 antibodies contribute to therapeutic development for inflammatory and fibrotic diseases?

CX3CR1 antibodies play crucial roles in therapeutic development through several research applications:

• Target validation: Antibodies against CX3CR1 and its ligand CX3CL1 have demonstrated efficacy in preclinical models of fibrotic disorders, validating this pathway as a therapeutic target. For example, anti-CX3CL1 monoclonal antibody treatment significantly reduced skin and lung fibrosis in sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) mouse models .

• Biomarker development: CX3CR1 antibodies enable quantification of receptor expression in patient samples, potentially identifying individuals more likely to respond to therapies targeting this pathway. Increased serum soluble CX3CL1 levels correlate with severe systemic sclerosis, suggesting utility as a stratification biomarker .

• Mechanism of action studies: Using CX3CR1 antibodies in histopathological analyses revealed that therapeutic effects of CX3CL1 blockade correlate with reduced infiltration of CX3CR1-expressing macrophages and T cells in affected tissues, providing mechanistic insights .

• Safety assessment: The absence of adverse events in preclinical studies with anti-CX3CL1 mAb treatment supports further exploration of this therapeutic approach .

• Companion diagnostic potential: CX3CR1 antibodies could potentially serve as tools for companion diagnostics to identify patients with elevated pathway activity.

Research data supports these applications, as anti-CX3CL1 mAb therapy has shown promise across multiple fibrosis models, suggesting broader utility in human fibrotic disorders .

What are the technical considerations for using CX3CR1 antibodies in human tissue samples for translational research?

Translational research using CX3CR1 antibodies in human samples requires careful technical considerations:

• Antibody selection:

  • Confirm human reactivity with validation data

  • Consider antibodies targeting conserved epitopes between species

  • Select antibodies with demonstrated performance in similar applications

  • Human/mouse cross-reactive antibodies (like AF5825) have been validated for both species

• Sample preparation:

  • Fresh frozen tissues generally yield better results than FFPE for membrane proteins like CX3CR1

  • For FFPE samples, optimize antigen retrieval protocols (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Consider section thickness (5-10 μm optimal for most applications)

  • Test multiple fixation protocols when possible

• Validation in human samples:

  • Always include known positive controls (e.g., NK cells, certain monocyte populations)

  • Perform peptide competition assays to confirm specificity

  • Compare staining patterns with published literature

  • Consider dual staining with antibodies against other established markers

• Interpretation challenges:

  • Account for variability in CX3CR1 expression levels between individuals

  • Consider effects of medications on receptor expression

  • Disease state may alter receptor internalization or shedding

  • Tissue processing delays can impact membrane protein detection

• Ethical and practical considerations:

  • Ensure appropriate ethical approvals and consent

  • Document clinical information relevant to interpretation

  • Consider time from collection to fixation as a variable

  • Standardize protocols across cohorts to enable comparison

Successful applications in human samples have been reported, including detection of CX3CR1 in oral squamous cell carcinoma cells and organoid cultures .

How is CX3CR1 expression being studied in the context of cancer immunology and metastasis?

CX3CR1 has emerged as a significant factor in cancer biology, with antibodies enabling several key research applications:

• Tumor microenvironment characterization: CX3CR1 antibodies help identify specific immune infiltrates in tumors, revealing the composition and potential function of myeloid and lymphoid populations. Recent research indicates CX3CR1 expression influences cell migration and invasion in oral squamous cell carcinoma through ICAM-1 expression .

• Metastatic process investigation: CX3CR1-expressing cells may facilitate tumor cell migration and invasion. Antibody-based detection systems allow tracking of CX3CR1+ cells in primary and metastatic sites.

• Therapeutic target assessment: As the CX3CL1-CX3CR1 axis influences tumor-immune interactions, antibodies enable validation of this pathway as a potential therapeutic target in cancer models.

• Prognostic biomarker evaluation: Quantification of CX3CR1+ cells in tumor samples may correlate with clinical outcomes, potentially serving as a prognostic indicator.

Methodological approaches include:

• Multiplex immunofluorescence to analyze CX3CR1+ cells in relation to other immune markers and tumor cells

• Flow cytometry to quantify CX3CR1 expression on circulating immune cells in cancer patients

• In vitro migration and invasion assays using CX3CR1 antibodies to block function

• Single-cell analysis combining CX3CR1 antibodies with other markers to identify specific cellular subsets

These applications have provided insights into cancer progression mechanisms involving CX3CR1-expressing cells .

What are the latest methodological advances in CX3CR1 detection for single-cell and spatial biology applications?

Recent technological advances have expanded CX3CR1 detection capabilities:

• Single-cell protein analysis:

  • Mass cytometry (CyTOF) incorporation of CX3CR1 antibodies enables high-dimensional analysis of dozens of markers simultaneously

  • Spectral flow cytometry allows more fluorophore combinations with CX3CR1 antibodies

  • Single-cell Western blot techniques can verify CX3CR1 expression at the individual cell level

• Spatial biology applications:

  • Multiplex immunofluorescence techniques combining CX3CR1 with multiple markers in single tissue sections

  • Imaging mass cytometry for high-parameter spatial analysis of CX3CR1+ cells

  • In situ hybridization combined with CX3CR1 immunostaining to correlate protein and mRNA expression

  • Proximity ligation assays to study CX3CR1 interactions with signaling partners

• Live cell imaging advances:

  • Fluorescently-tagged CX3CR1 antibody fragments for dynamic imaging

  • Calcium flux imaging combined with CX3CR1 staining to correlate expression with functional responses

  • Optogenetic approaches incorporating CX3CR1 targeting

• Multi-omics integration:

  • Antibody-based cell sorting followed by single-cell RNA sequencing

  • CITE-seq approaches incorporating CX3CR1 antibodies for simultaneous protein and transcriptome analysis

  • Spatial transcriptomics combined with CX3CR1 immunostaining for comprehensive tissue analysis

These methodological advances enable unprecedented insights into CX3CR1 biology in complex tissues and disease states, supporting deeper understanding of its role across biological systems .

What are the recommended resources for CX3CR1 antibody validation and protocol optimization?

For comprehensive CX3CR1 antibody information, researchers should consult:

• Validated antibody repositories:

  • Antibodypedia for independent validation data

  • The Antibody Registry for unique identifier tracking

  • RRID portal for antibody standardization

• Technical resources:

  • Manufacturer technical support (BiCell Scientific, R&D Systems, Abcam)

  • Application-specific protocols with optimization guidelines

  • Troubleshooting guides for common detection methods

• Literature resources:

  • Systematic reviews of CX3CR1 detection methods

  • Primary research articles demonstrating successful applications

  • Method papers describing optimized protocols

• Online communities and databases:

  • Research Gate forums for technical discussions

  • BioCompare for antibody comparison data

  • LinkedIn professional groups focused on antibody technologies

• Standardization initiatives:

  • International Working Group for Antibody Validation guidelines

  • Reproducibility initiatives like the Antibody Validation Initiative