CCR2 Antibody

What is the optimal sample preparation method for detecting CCR2 on monocytes via flow cytometry?

When detecting CCR2 on monocytes via flow cytometry, sequential staining procedures significantly improve detection quality. Evidence indicates that antibody panel design is critical, as certain antibody combinations can interfere with detection levels. Specifically, when simultaneously evaluating CCR2, CX3CR1, and CCR5, the ligation of CCR5 antibodies can interfere with detection of both CCR2 and CX3CR1 .

Recommended protocol:

• Isolate peripheral blood mononuclear cells (PBMCs) via density gradient centrifugation

• Follow a sequential staining procedure:

  • First stain with anti-CCR2 and anti-CX3CR1 antibodies

  • After washing, stain with anti-CCR5 antibodies

  • Include CD14 antibodies to identify monocyte populations

  • Use appropriate fluorescence-minus-one (FMO) controls

This approach helps overcome the detection issues caused by possible receptor proximity or functional interactions between CCR2/CCR5 and CX3CR1/CCR5 on monocytes .

What are the key technical differences between CCR2 antibody clones available for immunological research?

Different CCR2 antibody clones have distinct properties that affect their performance in various applications. Based on the available data, significant differences exist between common clones:

For studying human samples in flow cytometry applications, clone 48607 shows effective staining of blood monocytes and can be used in conjunction with CD14 markers . For murine models, EPR20844-15 offers broader application potential with KO-validation confirming specificity .

How can researchers optimize CCR2 antibody staining when conducting multiparameter flow cytometry for immune cell profiling?

Multiparameter flow cytometry with CCR2 antibodies requires careful optimization due to complex receptor interactions and internalization dynamics:

Critical optimization steps:

• Sequential staining approach: Add CCR2 antibodies before other chemokine receptor antibodies to prevent epitope masking or receptor internalization

• Temperature control: Maintain samples at 4°C during staining to minimize receptor internalization

• Panel design considerations:

  • Use Mouse Anti-Human CD14 Fluorescein-conjugated antibodies (e.g., FAB3832F) to identify monocyte populations

  • Apply Mouse Anti-Human CCR2 Biotinylated antibodies (e.g., FAB151B) with appropriate streptavidin conjugates for increased sensitivity

  • Include matching isotype controls (e.g., Mouse IgG Biotinylated Isotype Control IC0041B)

• Critical controls: Always include fluorescence-minus-one (FMO) controls when evaluating chemokine receptor co-expression to identify interference between antibodies

Implementing these approaches has been demonstrated to restore detection levels that are otherwise compromised when using simultaneous staining methods, particularly when assessing CCR2, CCR5, and CX3CR1 expression patterns .

What are the most effective approaches for validating CCR2 antibody specificity in different experimental contexts?

Validating CCR2 antibody specificity is crucial for generating reliable research data. Several approaches offer complementary validation strategies:

Knockout validation:

• Use CCR2 knockout cell lines as negative controls to confirm antibody specificity

• The EPR20844-15 clone has been validated using this approach, demonstrating absence of staining in CCR2 knockout samples

Multi-tissue microarray validation:

• Evaluate antibody performance across diverse tissue types

• Confirms both specificity and sensitivity in complex tissue microenvironments

• Look for expected staining patterns (e.g., lymphocyte staining in tonsil tissue)

Comparative detection methods:

• Parallel validation using orthogonal techniques (e.g., RNA-seq, mass spectrometry)

• Correlation between protein and mRNA expression levels

Application-specific validation:

• For flow cytometry: Compare staining between CCR2-expressing cells (e.g., CD14+ monocytes) and non-expressing populations

• For IHC: Human tonsil tissue shows specific CCR2 staining localized to lymphocytes when using the 48607 clone at 50 μg/mL (overnight at 4°C)

• For Western blot: Verify expected molecular weight (~42 kDa) and absence of non-specific bands

How should researchers design experiments to investigate CCR2's role in monocyte migration and recruitment in inflammation models?

When designing experiments to investigate CCR2's role in monocyte migration and inflammation, consider these methodological approaches:

In vitro migration assays:

• Transwell migration system:

  • Seed purified monocytes in upper chamber

  • Add CCL2/MCP-1 (primary CCR2 ligand) to lower chamber

  • Pre-treat cells with CCR2 antibodies (e.g., clone 48607) at varying concentrations to establish dose-dependent blockade

  • Quantify migration index relative to controls

Flow cytometry characterization:

• Identify CCR2+ monocyte subsets using standardized panels:

  • Human: CD14+ CD16- CCR2high (classical monocytes)

  • Mouse: Ly6Chigh CCR2high (inflammatory monocytes)

• Apply sequential staining protocol to avoid epitope interference

In vivo models:

• Adoptive transfer experiments:

  • Label CCR2+ monocytes with fluorescent dyes

  • Administer cells to recipient animals with localized inflammation

  • Use intravital microscopy to track migration patterns

  • Compare with parallel experiments using CCR2-blocked or CCR2-/- cells

Mechanistic analysis:

• Assess downstream signaling pathways:

  • PI3K cascade activation

  • Small G protein Rac engagement

  • Lamellipodium protrusion

These experimental approaches provide complementary data on CCR2 function, especially considering its key role in the PI3K signaling cascade and regulation of cellular migration in inflammatory contexts .

What considerations are important when using CCR2 antibodies for studying receptor internalization and trafficking dynamics?

CCR2 undergoes complex internalization and trafficking processes that require specialized experimental approaches:

Technical considerations for internalization studies:

• Temperature management:

  • Perform binding studies at 4°C to prevent internalization

  • Shift to 37°C to initiate internalization kinetics

  • Use time-course analyses with fixed timepoints to capture trafficking dynamics

• Detection strategies:

  • Surface vs. intracellular staining: Use permeabilization protocols to distinguish membrane-bound from internalized receptors

  • pH-sensitive fluorophores: Apply antibodies conjugated to pH-sensitive dyes that change emission characteristics when trafficking to acidic endosomal compartments

• Advanced microscopy approaches:

  • Confocal microscopy with z-stack imaging to confirm internalization

  • TIRF (Total Internal Reflection Fluorescence) microscopy to focus on membrane-proximal events

  • Live-cell imaging to track real-time receptor movement

• Controls and validation:

  • Use CCR2 antagonists to block ligand-induced internalization

  • Include dynamin inhibitors (e.g., Dynasore) to prevent endocytosis

  • Compare with constitutively recycling receptors as internal controls

Remember that even in the absence of ligand, CCR2 undergoes basal internalization, intracellular trafficking, and recycling to the cell surface, which may affect detection levels during extended experimental procedures .

What are the most common technical pitfalls when using CCR2 antibodies in flow cytometry, and how can they be overcome?

When using CCR2 antibodies in flow cytometry, researchers frequently encounter several technical challenges:

Common pitfalls and solutions:

• Reduced signal intensity due to receptor internalization:

  • Problem: CCR2 undergoes continuous internalization and recycling

  • Solution: Maintain samples at 4°C during processing and staining; use sodium azide in buffers to inhibit metabolic processes

• Antibody interference in multiplex panels:

  • Problem: Co-staining with CCR5 antibodies reduces CCR2 detection

  • Solution: Implement sequential staining protocols; add CCR2 antibodies first, followed by other chemokine receptor antibodies

• High background staining:

  • Problem: Non-specific binding to Fc receptors on monocytes

  • Solution: Include Fc receptor blocking reagents in staining buffer; optimize antibody concentration through titration

• Inconsistent results between experiments:

  • Problem: Variability in receptor expression due to sample handling

  • Solution: Standardize processing time; use consistent anticoagulants (EDTA preferred); process samples within 2 hours of collection

• Failed detection in activated cells:

  • Problem: Activation-induced receptor downregulation

  • Solution: Consider timing of staining relative to activation; include positive controls of non-activated cells

Validation workflow:

• Always include appropriate isotype controls (e.g., Mouse IgG for clone 48607)

• Use fluorescence-minus-one (FMO) controls to set accurate gates

• Include known positive populations (e.g., CD14+ monocytes) as internal standards

How can researchers accurately quantify CCR2 surface expression levels while accounting for receptor internalization dynamics?

Accurately quantifying CCR2 surface expression requires specialized approaches to account for receptor trafficking:

Methodological approaches:

• Kinetic analysis protocol:

  • Perform time-course measurements after ligand exposure

  • Plot surface expression relative to baseline at defined timepoints

  • Calculate internalization rates from slope of expression decline

• Quantitative flow cytometry:

  • Use antibody binding capacity (ABC) beads to convert fluorescence intensity to absolute receptor numbers

  • Apply calibration to convert mean fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF)

  • Calculate receptors per cell using standard curves

• Internalization-resistant antibody selection:

  • Choose non-competing antibody clones that maintain binding during receptor conformational changes

  • Use antibodies targeting receptor domains less affected by internalization

• Complementary techniques:

  • Surface biotinylation assays to track total surface protein fate

  • Fluorescence quenching assays to distinguish surface from internalized receptors

Standardization approaches:

• Always include biological standards with known CCR2 expression levels

• Normalize data to these standards across experiments

• Report both relative and absolute quantification metrics

This comprehensive approach helps researchers distinguish between altered receptor expression versus altered receptor localization in experimental systems .

How can researchers effectively use CCR2 antibodies to investigate its role in T-cell differentiation and inflammatory processes?

CCR2 plays critical roles in T-cell differentiation, particularly in promoting Th17 differentiation during inflammation. To investigate these functions:

Experimental approaches:

• T-cell subset analysis protocol:

  • Isolate CD4+ T cells from peripheral blood or tissues

  • Stain for CCR2 using validated antibodies (e.g., clone 48607)

  • Co-stain with markers for T-cell subsets (Th1, Th2, Th17, Treg)

  • Analyze correlation between CCR2 expression and specific T-cell phenotypes

• Functional assessment:

  • Sort CCR2+ vs. CCR2- T cells

  • Measure cytokine production profiles (IL-17, IFN-γ, IL-4)

  • Evaluate JAK-STAT pathway activation status

  • Assess FOXO1 activity and S1P1R expression levels

• Tissue-specific investigations:

  • Apply CCR2 antibodies in IHC of inflamed tissues (e.g., human tonsil)

  • Use the anti-CCR2 clone 48607 at 50 μg/mL overnight at 4°C

  • Detect with HRP-DAB system for chromogenic visualization

  • Counterstain with hematoxylin to identify tissue architecture

• Co-expression studies:

  • Identify CCR2+CCR5+ T cell populations, which have been shown to produce Matrix Metalloproteinase-9 and Osteopontin in multiple sclerosis pathogenesis

  • Evaluate their specific contributions to inflammatory processes

This methodological framework enables researchers to characterize how CCR2-expressing T cells contribute to inflammatory conditions and autoimmune pathologies.

What are the best approaches for studying CCR2's role in pathological conditions using tissue-based imaging techniques?

Investigating CCR2's role in pathological conditions through tissue imaging requires specialized techniques:

Methodological considerations:

• Tissue preparation optimization:

  • For paraffin-embedded sections: Use antigen retrieval methods (citrate buffer, pH 6.0)

  • For frozen sections: Brief fixation (2-4% PFA, 10 minutes) preserves epitope accessibility

  • Section thickness: 5-7 μm optimal for CCR2 detection

• Staining protocol for optimal detection:

  • Apply validated anti-CCR2 antibodies (e.g., clone 48607) at 50 μg/mL

  • Incubate overnight at 4°C for maximum sensitivity

  • Use HRP-DAB detection systems for chromogenic visualization

  • Consider tyramide signal amplification for low-expressing tissues

• Multiplex immunofluorescence approaches:

  • Combine CCR2 detection with cell-type markers (CD14, CD3, CD68)

  • Add tissue context markers (CD31 for vasculature, Collagen IV for basement membranes)

  • Use sequential staining to prevent antibody cross-reactivity

  • Apply spectral unmixing for closely related fluorophores

• Quantitative analysis methods:

  • Whole slide imaging with automated quantification

  • Cell-by-cell analysis of CCR2 expression intensity

  • Spatial relationship mapping between CCR2+ cells and tissue structures

  • Correlation with clinical parameters and outcomes

Pathology-specific considerations:

• In neuroinflammatory conditions: Co-stain with neural markers to assess CCR2's role in mediating peripheral nerve injury-induced neuropathic pain

• In tumor microenvironment: Evaluate CCR2+ inflammatory monocyte infiltration patterns

• In autoimmune diseases: Assess relationship between CCR2+ cells and tissue damage

These approaches enable comprehensive characterization of how CCR2-expressing cells contribute to tissue pathology in various disease states.

How can researchers effectively combine CCR2 antibodies with other technologies for comprehensive immune cell profiling?

Integrating CCR2 antibody staining with emerging technologies enhances immune cell characterization:

Cutting-edge methodological approaches:

• Single-cell RNA sequencing integration:

  • Sort CCR2+ and CCR2- populations using validated antibodies

  • Perform scRNA-seq to identify transcriptional signatures

  • Correlate surface CCR2 protein levels with mRNA expression

  • Identify novel genes co-regulated with CCR2 in specific cell subsets

• Mass cytometry (CyTOF) implementation:

  • Use metal-conjugated CCR2 antibodies in comprehensive immune panels

  • Combine with 30+ other markers for high-dimensional phenotyping

  • Apply unsupervised clustering to identify novel CCR2+ populations

  • Validate findings through conventional flow cytometry

• Spatial transcriptomics correlation:

  • Perform CCR2 immunostaining on serial tissue sections

  • Correlate with spatial transcriptomics data

  • Map CCR2+ cell distributions relative to tissue microenvironments

  • Identify spatial relationships between CCR2+ cells and other immune populations

• Functional metabolic assessment:

  • Isolate CCR2+ cells using antibody-based magnetic separation

  • Perform metabolic profiling (Seahorse assays)

  • Correlate CCR2 expression with metabolic states

  • Link receptor expression to functional cellular programs

These integrated approaches provide multi-dimensional insights into CCR2 biology beyond traditional single-parameter analyses.

What methodological approaches are most effective for investigating the interplay between CCR2 and other chemokine receptors in complex immune responses?

The functional interplay between CCR2 and other chemokine receptors requires specialized experimental approaches:

Advanced methodological strategies:

• Receptor co-expression analysis protocol:

  • Implement optimized sequential staining for flow cytometry

  • First add CCR2 antibodies, followed by antibodies against CX3CR1, CCR5, and other chemokine receptors

  • Include proper FMO controls to identify potential interference

  • Analyze co-expression patterns at single-cell resolution

• Competitive binding assays:

  • Pre-incubate cells with unlabeled antibodies against one receptor

  • Follow with labeled antibodies against second receptor

  • Quantify binding alterations to assess receptor proximity or dimerization

  • Perform in both directions to confirm interaction specificity

• Functional migration studies:

  • Design transwell assays with combined chemokine gradients

  • Assess synergistic or antagonistic effects on migration

  • Block individual receptors with specific antibodies to determine hierarchical contributions

  • Correlate with receptor surface expression patterns

• Proximity ligation assays:

  • Apply antibodies against CCR2 and other chemokine receptors

  • Use species-specific secondary antibodies with DNA probes

  • Visualize receptor proximity through amplified fluorescent signal

  • Quantify interaction events per cell using microscopy

These approaches help elucidate the functional consequences of the close proximity and possible interactions observed between CCR2/CCR5 and CX3CR1/CCR5, which have significant implications for cellular migration and inflammatory responses .